Methods and systems for processing biomass material

ABSTRACT

Embodiments of the present invention provide for efficient and economical production and recovery of volatile organic compounds and hydrocarbons. One embodiment comprises contacting a solid component of a biomass material with a solution adapted to facilitate saccharification, and contacting the at least one fermentable sugar with a microorganism capable of using the at least one fermentable sugar to generate a hydrocarbon. The solid component is generated by introducing a biomass material to a compartment of a solventless recovery system, wherein the biomass material contains one or more volatile organic compounds; contacting the biomass material with a superheated vapor stream in the compartment to vaporize at least a portion of an initial liquid content in the biomass material; separating a vapor component and a solid component from the heated biomass material; and retaining at least a portion of the gas component for use as part of the superheated vapor stream.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/648,109, filed on May 17, 2012, U.S. Provisional Application No.61/786,844, filed on Mar. 15, 2013, and U.S. Provisional Application No.61/786,860, filed on Mar. 15, 2013, the disclosures of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

Embodiments of this invention relate generally to processing of biomassmaterial and more particularly to manufacturing and recovery of volatileorganic compounds and hydrocarbon compounds, using readily availablefermentable sugar and fermentable sugar from further processing oflignocellulosic material in the biomass material.

BACKGROUND

This section is intended to introduce various aspects of the art, whichmay be associated with exemplary embodiments of the present invention.This discussion is believed to assist in providing a framework tofacilitate a better understanding of particular aspects of the presentinvention. Accordingly, it should be understood that this section shouldbe read in this light, and not necessarily as admissions of any priorart.

As the world's petroleum supplies continue to diminish there is agrowing need for alternative materials that can be substituted forvarious petroleum products, particularly transportation fuels. Asignificant amount of effort has been placed on developing new methodsand systems for providing energy from resources other than fossil fuels.Currently, much effort is underway to produce bioethanol and othertransportation fuels and chemicals from renewable biomass materials. Onetype of biomass is plant biomass, which contains a high amount ofcarbohydrates including sugars, starches, celluloses, lignocelluloses,hemicelluloses. Efforts have particularly been focused on ethanol fromfermentable sugar readily available and ethanol from cellulosicmaterials.

Conventional ethanol production from corn using readily availablefermentable sugar typically competes with valuable food resources, whichcan be further amplified by increasingly more severe climate conditions,such as droughts and floods, which negatively impact the amount of cropharvested every year. The competition from conventional ethanolproduction can drive up food prices. While other crops have served asthe biomass material for ethanol production, they usually are notsuitable for global implementations due to the climate requirements ofsuch crops. For instance, ethanol can also be efficiently produced fromsugar cane, but only in certain areas of the world, such as Brazil, thathave a climate that can support near-year-round harvest.

Further, additional fermentable sugars can be freed from lignocellulosicbiomass, which comprises hemicelluloses, cellulose and smaller portionsof lignin and protein. Cellulose comprises sugars that can be convertedinto fuels and valuable chemicals, when they are liberated from the cellwalls and polymers that contain them.

Current processes aiming to process lignocellulosic biomass are limitedto feedstock that includes unprocessed biomass materials or municipalsolid waste (MSW). Unprocessed biomass includes sugarcane bagasse,forest resources, crop residues, and wet/dry harvested energy crops.These conventional feedstock sources require storage, transportation,particle size reduction, and additional front end processing before theycan be introduced for further processing of lignocellulosic material.For example, baling of biomass is costly and can result in hazards suchas fire, rodent, dust, unwanted debris (such as rocks) and hantavirus.Further, bales and forest resources are more costly to transport thandenser material and more costly to handle than materials that arealready particle size reduced and do not need to be further formatted.MSW further has challenges related to contamination with regulatedhazardous metals that can contribute to risks of poor fuel quality aswell as health and safety risks. Forest resources, such as trees, arecumbersome to transport. Further, forest resources require debarking,chopping to wood chips of desirable thickness, and washing to remove anyresidual soil, dirt and the like. Therefore, there is still a need for abiomass that addresses these challenges.

SUMMARY

Embodiments of the invention can address the challenges mentioned aboveas well as provide other advantages and features. In one embodiment, thefeedstock can come from the solid component exiting a volatile organiccompound recovery system. In that embodiment, the feedstock is alreadyflowable in an engineered system, which allows the feedstock to berouted directly into the reactor to generate additional fermentablesugars as desired. Embodiments of the invention can provide for avolatile organic compound recovery equipment to recover products fromthe fermentation phase and further processing of lignocellulosicmaterial equipment to be located near each other. The further processingcan yield additional fermentable sugar that can be converted tohydrocarbons and/or other chemicals. Such embodiments can allow forproduction of volatile organic compounds from fermentation and furtherprocessing of lignocellulosic material, which reduces storage, handling,and transportation costs associated with other feedstock before it canenter the production flow of the further processing of lignocellulosicmaterial. Such embodiments can also provide a continuous supply offeedstock that is already formatted in contrast to conventionalfeedstock that often requires storage, transportation, and/or formattingat or prior to arriving at the biomass facility for processing of thelignocellulosic material, which reduces the particular associated costs.

The feedstock of certain embodiments can also have lower handling andtransportation costs when it is transported to other locations forprocessing of the lignocellulosic material. Unlike other conventionalfeedstock sources, such as forest resources, the feedstock of certainembodiments exits the volatile organic compound recovery system in apreformatted manner that is particle-size reduced, which can reduce oreliminate the front end processing costs before the feedstock can enterthe processing of lignocellulosic material. The preformatted sizedistribution of the feedstock of certain embodiments of the inventionplaces it in a denser form than other conventional feedstock sources,which can reduce transportation cost as more of the feedstock of theseembodiments can be transported per volume. Embodiments of the inventioncan provide a supply of feedstock that is available year-roundindependent of a harvest period particular to a biomass material thusreducing storage needs and costs for the further processing plant anddoes not compete with valuable food sources for human.

In one embodiment, a biomass material is prepared to generate volatileorganic compounds. The volatile organic compounds are recovered from theprepared biomass material by introducing the prepared biomass materialto a compartment of a solventless recovery system; contacting thebiomass material with a superheated vapor stream in the compartment tovaporize at least a portion of an initial liquid content in the preparedbiomass material, the superheated vapor stream comprising at least onevolatile organic compound; separating a vapor component and a solidcomponent from the heated biomass material, where the vapor componentcomprises at least one volatile organic compound; and retaining at leasta portion of the gas component for use as part of the superheated vaporstream. Compounds in the vapor component can be further purified throughan appropriate distillation process. At least a portion of the solidcomponent is further processed to generate additional fermentablesugars. In one embodiment, the further processing comprises contactingat least a portion of the solid component with a solution adapted tofacilitate saccharification to generate additional fermentable sugars.In one embodiment, the additionally generated fermentable sugars areconverted to at least one hydrocarbon compound through fermentationusing one or more microorganism.

In one embodiment, the one or more organism is a recombinant host cell(or microorganism) adapted to produce a hydrocarbon as disclosed by PCTApplication No. PCT/EP2013/053600, the disclosure of which isincorporated by reference in its entirety. For example, in oneembodiment, the recombinant host cell is adapted to express at least oneof a fatty acid reductase, a fatty aldehyde synthetase, a fatty acyltransferase, and a aldehyde decarbonylase, where at least one fatty acidreductase, at least one fatty aldehyde synthetase, and at least onefatty acyl transferase forms a fatty acid reductase complex. Contact ofa fatty acid substrate with a fatty aldehyde synthetase forms a fattyacid aldehyde. Contact of the fatty acid aldehyde with at least onealdehyde decarbonylase forms a hydrocarbon.

The fatty acid substrate may be a fatty acid, a fatty acyl-ACP (fattyacyl-acyl carrier protein) or fatty acyl-CoA or a mixture of any ofthese. The fatty acid reductase complex may comprise a fatty acidreductase enzyme polypeptide having Enzyme Commission (EC) no. 1.2.1.50,for example having at least 50% sequence identity with SEQ ID NO:1(Photorhabdus luminescens protein LuxC). Additionally or independently,the fatty acid reductase complex may comprise a fatty aldehydesynthetase enzyme polypeptide having EC no. 6.2.1.19, for example havingat least 50% sequence identity with SEQ ID NO:2 (P. luminescens proteinLuxE). Additionally or independently, the fatty acid reductase complexmay comprise a fatty acyl transferase enzyme polypeptide in class EC2.3.1.-, for example having at least 50% sequence identity to SEQ IDNO:3 (P. luminescens protein LuxD). Additionally or independently, thealdehyde decarbonylase may be in class EC 4.1.99.5, for example it maybe a polypeptide having at least 50% sequence identity with SEQ ID NO:4(Nostoc punctiforme aldehyde decarbonylase protein). In an exemplaryembodiment, all of the enzymes having the sequences SEQ ID NOs:1-4 areused.

In one embodiment, the prepared biomass is generated by adding to thebiomass at least one additive added, wherein said at least one additivecomprise a microbe, and optionally, an acid and/or an enzyme; andstoring the prepared biomass material for at least about 24 hours in astorage facility to allow for the production of at least one volatileorganic compound from at least a portion of the sugar.

In addition to the features described above, embodiments of theinvention allow for economical production of alternative fuels, such asethanol, other volatile organic compounds, hydrocarbons, and otherchemicals, from plants that contain fermentable sugar by addressingchallenges, such as costs of storage and transportation, short harvestwindows, quick degradation of sugars, and large investment in equipment.Aspects of the embodiments described herein are applicable to anybiomass material, such as plants containing fermentable sugars. Thefeatures of embodiments of the present invention allow for economicaluse of various plants to produce alternative fuels and chemicals and arenot limited to sorghum and other plants that suffer similar challenges.Such challenging crops are highlighted herein because other methods andsystems have not been able to economically use these challenging cropsto produce fuels and chemicals. As such, the specific mention of sorghumis not intended to be limiting, but rather illustrates one particularapplication of embodiments of the invention.

Embodiments of the invention allow for the recovery facility to runcontinuously year-round in a controlled manner independent of theharvest window, thereby broadening the geological locations available toplace a recovery facility and/or a facility to process lignocellulosicmaterial, including areas with a relatively short harvest window.

Other advantages and features of embodiments of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

These drawings illustrate certain aspects of some of the embodiments ofthe invention, and should not be used to limit or define the invention.

FIG. 1 is a diagram of one embodiment to process biomass materialaccording to certain aspects of the present invention.

FIG. 2 is a diagram of another embodiment to process biomass materialaccording to certain aspects of the present invention.

FIG. 3 is a diagram of a particular embodiment for saccharification of asolid component according to aspects of the invention.

FIG. 4 is a schematic detailing the genetic elements (solid lines)introduced into E. coli cells according to certain aspects of theinvention;

FIG. 5 shows the growth curve for various E. coli cultures obtainedaccording to certain aspects of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the present invention can provide efficient andeconomical production and recovery of ethanol or other volatile organiccompounds, such as ethanol and acetic acid, from solid biomass material,as well as a feedstock for further processing of lignocellulosicmaterial to generate fermentable sugar for conversion furtherfermentation, including production of hydrocarbon compounds. Accordingto one aspect of the invention, a biomass material is prepared togenerate volatile organic compounds. The volatile organic compounds arerecovered from the prepared biomass material by introducing the preparedbiomass material to a compartment of a solventless recovery system;contacting the biomass material with a superheated vapor stream in thecompartment to vaporize at least a portion of an initial liquid contentin the prepared biomass material, the superheated vapor streamcomprising at least one volatile organic compound; separating a vaporcomponent and a solid component from the heated biomass material, wherethe vapor component comprises at least one volatile organic compound;and retaining at least a portion of the gas component for use as part ofthe superheated vapor stream. At least a portion of the solid componentis further processed to generate additional fermentable sugar. In oneembodiment, the further processing contacting at least a portion of thesolid component with a solution adapted to facilitate saccharification.In one embodiment, the additionally generated fermentable sugars arefermented to produce a hydrocarbon.

Biomass Preparation

As used herein, the term “solid biomass” or “biomass” refers at least tobiological matter from living, or recently living organisms. Solidbiomass includes plant or animal matter that can be converted intofibers or other industrial chemicals, including biofuels. Solid biomasscan be derived from numerous types of plants or trees, includingmiscanthus, switchgrass, hemp, corn, tropical poplar, willow, sorghum,sugarcane, sugar beet, and any energy cane, and a variety of treespecies, ranging from eucalyptus to oil palm (palm oil). In oneembodiment, the solid biomass comprises at least one fermentablesugar-producing plant. The solid biomass can comprise two or moredifferent plant types, including fermentable sugar-producing plant. In apreferred embodiment not intended to limit the scope of the invention,sorghum is selected, due to its high-yield on less productive lands andhigh sugar content.

The term “fermentable sugar” refers to oligosaccharides andmonosaccharides that can be used as a carbon source (e.g., pentoses andhexoses) by a microorganism to produce an organic product such asalcohols, organic acids, esters, and aldehydes, under anaerobic and/oraerobic conditions. Such production of an organic product can bereferred to generally as fermentation. The at least one fermentablesugar-producing plant contains fermentable sugars dissolved in the waterphase of the plant material at one point in time during its growthcycle. Non-limiting examples of fermentable sugar-producing plantsinclude sorghum, sugarcane, sugar beet, and energy cane. In particular,sugarcane, energy cane, and sorghum typically contain from about 5% toabout 25% soluble sugar w/w in the water phase and have moisture contentbetween about 60% and about 80% on a wet basis when they are near or attheir maximum potential fermentable sugar production (e.g., maximumfermentable sugar concentration).

The term “wet basis” refers at least to the mass percentage thatincludes water as part of the mass. In a preferred embodiment, the sugarproducing plant is sorghum. Any species or variety of the genus sorghumthat provides for the microbial conversion of carbohydrates to volatileorganic compounds (VOCs) can be used. For embodiments using sorghum, theplant provides certain benefits, including being water-efficient, aswell as drought and heat-tolerant. These properties make the cropsuitable for many locations, including various regions across the earth,such as China, Africa, Australia, and in the US, such as portions of theHigh Plains, the West, and across the South. Texas.

In embodiments using sorghum, the sorghum can include any variety orcombination of varieties that may be harvested with higherconcentrations of fermentable sugar. Certain varieties of sorghum withpreferred properties are sometimes referred to as “sweet sorghum.” Thesorghum can include a variety that may or may not contain enoughmoisture to support the juicing process in a sugar cane mill operation.In a preferred embodiment, the solid biomass includes a Sugar T sorghumvariety commercially produced by Advanta and/or a male parent of SugarT, which is also a commercially available product of Advanta. In apreferred embodiment, the crop used has from about 5 to about 25 brix,preferably from about 10 to about 20 brix, and more preferably fromabout 12 to about 18 brix. The term “brix” herein refers at least to thecontent of glucose, fructose, and sucrose in an aqueous solution whereone degree brix is 1 gram of glucose, fructose, and/or sucrose in 100grams of solution and represents the strength of the solution aspercentage by weight (% w/w). In another preferred embodiment, themoisture content of the crop used is from about 50% to 80%, preferablyat least 60%.

In one embodiment, the crop is a male parent of Sugar T with a brixvalue of about 18 and a moisture content of about 67%. In anotherembodiment, the crop is Sugar T with a brix value of about 12 at amoisture content of about 73%. In these particular embodiments, the brixand moisture content values were determined by handheld refractometer.

After at least one additive (a microbe, optionally, an acid and/orenzyme) is added to the solid biomass, it becomes prepared biomassmaterial where the at least one additive facilitates the conversion offermentable sugar into a VOC (such as ethanol). As noted above andfurther described below, the prepared biomass material can be stored fora certain period of time to allow more VOCs to be generated by theconversion process. At least one volatile organic compound is thenrecovered from the prepared biomass material. Volatile organic compoundsare known to those skilled in the art. The U.S. EPA providesdescriptions volatile organic compounds (VOC), one of which is anycompound of carbon, excluding carbon monoxide, carbon dioxide, carbonicacid, metallic carbides or carbonates, and ammonium carbonate, whichparticipates in atmospheric photochemical reactions, except thosedesignated by EPA as having negligible photochemical reactivity (seehttp://www.epa.gov/iaq/voc2.html#definition). Another description ofvolatile organic compounds, or VOCs, is any organic chemical compoundwhose composition makes it possible for them to evaporate under normalindoor atmospheric conditions of temperature and pressure. This is thegeneral definition of VOCs that is used in the scientific literature,and is consistent with the definition used for indoor air quality.Normal indoor atmospheric conditions of temperature and pressure referto the range of conditions usually found in buildings occupied bypeople, and thus can vary depending on the type of building and itsgeographic location. One exemplary normal indoor atmospheric conditionis provided by the International Union of Pure and Applied Chemistry(IUPAC) and the National Institute of Standards and Technology (NIST).IUPAC's standard is a temperature of 0° C. (273, 15 K, 32° F.) and anabsolute pressure of 100 kPa (14.504 psi), and NIST's definition is atemperature of 20° C. (293, 15 K, 68° F.) and an absolute pressure of101.325 kPa (14.696 psi).

Since the volatility of a compound is generally higher the lower itsboiling point temperature, the volatility of organic compounds aresometimes defined and classified by their boiling points. Accordingly, aVOC can be described by its boiling point. A VOC is any organic compoundhaving a boiling point range of about 50 degrees C. to 260 degrees C.measured at a standard atmospheric pressure of about 101.3 kPa. Manyvolatile organic compounds that can be recovered and/or furtherprocessed from VOCs recovered from embodiments of the present inventionhave applications in the perfume and flavoring industries. Examples ofsuch compounds may be esters, ketones, alcohols, aldehydes, hydrocarbonsand terpenes. The following Table 1 further provides non-limitingexamples of volatile organic compounds that may be recovered and/orfurther processed from VOCs recovered from the prepared biomassmaterial.

TABLE 1 Methanol Ethyl acetate Acetaldehyde Diacetyl 2,3-pentanedioneMalic acid Pyruvic acid Succinic acid Butyric acid Formic acid Aceticacid Propionic acid Isobutyric acid Valeric acid Isovaleric acid2-methylbutyric acid Hexanoic acid Heptanoic acid Octanoic acid Nonanoicacid Decanoic acid Propanol Isopropanol Butanol Isobutanol IsoamylHexanol Tyrosol Tryptoptanol alcohol 2,3-butanediol Glycerol Fumaricacid Phenethyl Amyl alcohol 1,2-propanol 1-propanol alcohol Methylacetate Ethyl acetate Propyl acetate Ethanol Propyl lactate AcetoneEthyl formate 2-butanol 2-methyl-1- 2-propen-1-ol 2,3-methyl-1- Ethyllactate propanol butanol n-propyl alcohol 3-buten-2-ol

Ethanol is a preferred volatile organic compound. As such, many examplesspecifically mention ethanol. This specific mention, however, is notintended to limit the invention. It should be understood that aspects ofthe invention also equally apply to other volatile organic compounds.Another preferred volatile organic compound is acetic acid.

Embodiments of the present invention provide for the long term storageof solid biomass material without significant degradation to thevolatile organic compounds contained in the prepared biomass material,and they provide for sugar preservation to allow for continuedgeneration of VOCs. As used in this context, “significant” refers atleast to within the margin of error when measuring the amount orconcentration of the volatile organic compounds in the prepared biomassmaterial. In one embodiment, the margin of error is about 0.5%.

Accordingly, embodiments of the present invention allow for continuousproduction VOCs without dependence on the length of the harvest, therebyeliminating or minimizing down time of a recovery plant in traditionaljust-in-time harvest and recovery processes. As such, embodiments of thepresent invention allow for harvest of the crop at its peak withoutcompromises typically made to lengthen the harvest season, such asharvest slightly earlier and later than peak time. That is, embodimentsof the invention allow for harvest at high field yields and high sugarconcentrations, such as when the selected crop has reached its peaksugar concentration or amount of fermentable sugars that can beconverted into a volatile organic compound, even if this results in ashorter harvest period. In one embodiment, the solid biomass isharvested or prepared when it is at about 80%, about 85%, about 90%,about 95%, or about 100% of its maximum potential fermentable sugarconcentration. As such, embodiments of the present invention,particularly the recovery phase, can be operated continuously year-roundwithout time pressure from fear of spoilage of the solid biomass andVOCs contained therein. While embodiments of the present invention allowfor harvest of the solid biomass near or at its maximum sugar productionpotential, the solid biomass material can be harvested at any point whenit is deemed to contain a suitable amount of sugar. Further, the harvestwindow varies depending on the type of crop and the geographicallocation. For example, the harvest window for sorghum in North Americacan range from about 1 to 7 months. However, in Brazil and otherequatorial and near equatorial areas, the harvest window may be up totwelve months.

In embodiments using plants as the solid biomass, the solid biomass canbe collected or harvested from the field using any suitable means knownto those skilled in the art. In one embodiment, the solid biomasscomprises a stalk component and a leaf component of the plant. Inanother embodiment, the solid biomass further comprises a graincomponent. In a preferred embodiment, the solid biomass is harvestedwith a forage or silage harvester (a forage or silage chopper). A silageor forage harvester refers to farm equipment used to make silage, whichis grass, corn or other plant that has been chopped into small pieces,and compacted together in a storage silo, silage bunker, or in silagebags. A silage or forage harvester has a cutting mechanism, such aseither a drum (cutterhead) or a flywheel with a number of knives fixedto it, which chops and transfers the chopped material into a receptaclethat is either connected to the harvester or to another vehicle drivingalongside. A forage harvester is preferred because it provides benefitsover a sugar cane harvester or dry baled system. For example, a forageharvester provides higher density material than a sugar cane harvester,thereby allowing for more efficient transportation of the harvestedmaterial. In one embodiment, using a forage harvester results inharvested sorghum with a bulk density of about 400 kg/m³, compared tosugarcane harvested with a sugarcane harvester with density of about 300kg/m³, and for sorghum harvested with a sugarcane harvester with adensity of about 200 kg/m³. In general, higher bulk density material ischeaper to transport, which tends to limit the geographical area inwhich cane-harvested crop can be sourced.

Thus, a forage harvester is an overall less expensive way to harvest theselected biomass, such as sorghum, than a cane harvester or dry baledsystem. Not to be bound by theory, it is believed the cost savings aredue in part to higher material throughputs and the higher bulk densityof the solid biomass harvested by a forage harvester. The solid biomasscan be cut in any length. In one embodiment, the chop lengths of theharvester is set to a range of about 3 mm to about 80 mm, preferablyabout 3 mm to about 20 mm, with examples of about 3 mm to about 13 mmchop lengths being most preferred. At these preferred chop lengths,there was not observable aqueous discharge in the forage harvester, solosses were minimal. When a chop length is selected, the harvesterprovides biomass with an average size or length distribution of aboutthe chop length selected. In one embodiment, the average sizedistribution of the solid component exiting the recovery system can beadjusted as desired, which can be done by adjusting the chop length ofthe harvester.

At least one additive is added to the solid biomass to facilitate and/orexpedite the conversion of appropriate carbohydrates into volatileorganic compounds. After selected additive(s) have been added, the solidbiomass can be referred to as prepared biomass material. In oneembodiment, the prepared biomass material can comprise at least one orany combination of fermentable sugar-producing plants listed above. In apreferred embodiment, the selected additive(s) can be conveniently addedusing the harvester during harvest.

In one embodiment, at least about 700 tons, preferably at least about 1million tons, such as at least 1.2 million tons, or more preferablyabout at least 5 million tons of prepared biomass material is generatedin a particular harvest window based on the growing conditions of aspecific region, such as about 1 to 7 months in North America forsorghum.

The at least one additive can be added at any point during and/or afterthe harvest process. In a preferred embodiment using a forage harvester,additives are added to the solid biomass during the harvest process togenerate a prepared biomass material. In particular, forage harvestersare designed for efficiently adding both solid and liquid additivesduring harvest. As mentioned above, the additives added include at leasta microbe (e.g. a yeast), and optionally, an acid and/or an enzyme. In apreferred embodiment, the selected additive(s) are added as solutions.Additional details of the potential additives are further providedbelow.

For embodiments using a forage harvester or a similar equipment, theselected additive(s) can be added during harvest at all phases, such asbefore the intake feed rollers, during intake, at chopping, afterchopping, through the blower, after the blower, in the accelerator, inthe boom (or spout), and/or after the boom. In one embodiment where acidand enzyme are added, the acid is added near the intake feed rollers,and a microbe and the enzyme are added in the boom. In a particularembodiment, a Krone Big X forage harvester with a V12 motor with anabout 30 ft wide header is used. In an embodiment using the Kronesystem, the acid is added as a solution through flexible tubing thatdischarged the solution just in front of the feed rollers. In this way,the liquid flow can be visually monitored, which showed the acidsolution and solid biomass quickly mixed inside the chopping chamber. Inanother embodiment, the addition of acid was also demonstrated as aviable practice using a Case New Holland FX 58 forage harvester. Incertain embodiments, the forage harvester used can include an onboardrack for containing additives, at least the one(s) selected to be addedduring harvest. In another embodiment, the selected additive(s) to beadded during harvest may be towed behind the harvester on a trailer. Forexample, in one embodiment, it was demonstrated that a modified utilitytrailer equipped with tanks containing additive solutions of yeast,enzymes and acid can be employed with minimal interfering with normaloperations of the harvester, thereby substantially maintaining theexpected cost and duration of the harvest process. For example, a normalharvest configuration and biomass yield employing a silage harvestertravelling at about 4 miles per hour maintains a similar rate ofcollection of about 4 miles per hour when equipped with certainadditives as described above in one embodiment.

In embodiments of the present invention, the prepared biomass materialis eventually transported to a storage facility where it is stored for aperiod of time to allow for production of at least one volatile organiccompound from at least a portion of the fermentable sugar of the solidbiomass. The details of the storage phase are further provided below. Incertain embodiments, selected additive(s) can also be added at thestorage facility. For example, in one embodiment, the selectedadditive(s) can be added during unloading or after the solid biomass hasbeen unloaded at the storage facility. In one embodiment, a conveyancesystem is used to assist with the adding of selected additive(s) at thestorage facility. Additive(s) added at the storage facility to solidbiomass can be one(s) that have not been added or additional amount ofone(s) previously added. Accordingly, selected additive(s) can thereforebe added at any point from the start of the harvest process to prior tostorage of the prepared biomass material at the storage area orfacility, such as at points where the material is transferred.

As mentioned above, additive(s) for embodiments of the present inventioninclude at least a microbe and optionally, an acid and/or an enzyme.Selected additive(s) can be added to the solid biomass in any order. Ina preferred embodiment, an acid is added to the solid biomass beforeadding a microbe to prime the material to provide an attractive growthenvironment for the microbe.

In a preferred embodiment, acid is added to reduce the pH of the solidbiomass to a range that facilitates and/or expedites selected indigenousor added microbial growth, which increases production of ethanol and/orvolatile organic compounds. The acid can also stop or slow plantrespiration, which consumes fermentable sugars intended for subsequentVOC production. In one embodiment, acid is added until the pH of thesolid biomass is between about 2.5 and about 5.0, preferably in a rangeof about 3.7 to about 4.3, and more preferably about 4.2. The acid usedcan include known acids, such as sulfuric acid, formic acid, orphosphoric acid. The following Table 2 provides non-limiting examples ofan acid that can be used individually or in combination.

TABLE 2 Sulfuric Acid Formic Acid Propionic Acid Malic Acid PhosphoricAcid Maleic Acid Folic Acid Citric Acid

In a preferred embodiment, after the solid biomass has reached thedesired pH with the addition of acid, a microbe is added. A microbe inthe additive context refers at least to a living organism added to thesolid biomass that is capable of impacting or affecting the preparedbiomass material. One exemplary impact or effect from added microbe(s)includes providing fermentation or other metabolism to convertfermentable sugars from various sources, including cellulosic material,into ethanol or other volatile organic compounds. Another exemplaryimpact or effect may be production of certain enzyme(s) that help todeconstruct cellulose in the prepared biomass material into fermentablesugars which can be metabolized to ethanol or other VOC. Yet anotherexemplary impact or effect provided by a microbe includes production ofcompounds such as vitamins, co-factors, and proteins that can improvethe quality, and thus value, of an eventual by-product that can serve asfeed for animals. Further, microbial activity provides heat for thepile. Parts of the microbial cell walls or other catabolite or anabolitemay also offer value-added chemicals that may be recovered by a recoveryunit. These impacts and effects may also be provided by microbesindigenous to the solid biomass.

Any microbe that is capable of impacting or affecting the preparedbiomass material can be added. In a preferred embodiment, the microbe(s)can include microbes used in the silage, animal feed, wine, andindustrial ethanol fermentation applications. In one embodiment, themicrobe selected includes yeast, fungi, and bacteria according toapplication and the desired profile of the organic molecule to be made.In a preferred embodiment, yeast is the selected microbe. In anotherembodiment, bacteria can be added to make lactic acid or acetic acid.Certain fungi can also be added to make these acids. For example,Acetobacterium acetii can be added to generate acetic acid;Lactobacillus, Streptococcus thermophilus can be added to generatelactic acid; Actinobacillus succinogenes, Mannheimia succiniciproducens,and/or Anaerobiospirillum succiniciproducens can be added to generatesuccinic acid; Clostridium acetobutylicum can be added to generateacetone and butanol; and/or Aerobacter aerogenes can be added togenerate butanediol.

The following Table 3 provides non-limiting examples of preferredmicrobes, which can be used individually or in combination.

TABLE 3 Saccharomyces Saccharomyces Saccharomyces Saccharomycesfermentatti cerevisiae japonicas bayanus Saccharomyces SaccharomycesClostridium Clostridium exiguous chevalieri acetobutylicumamylosaccharobutylpropylicum Clostridium Clostridium ClostridiumAerobacter species propyl- viscifaciens propionicum butylicum AerobacterZymomonas Zymomonas Clostridium species aerogenes mobilis speciesSaccharomyces Bacillus species Clostridium Lactobacillus buchnerispecies thermocellum Lactobacillus Enterococcus PediococcusPropionibacteria plantarum faecium species Acetobacterium StreptococcusLactobacillus Lactobacillus species acetii thermophilus paracaseiActinobacillus Mannheimia Anaerobiospirillum succinogenessucciniciproducens succiniciproducens

Preferred microbes also include Saccharomyces cerevisiae strains thatcan tolerate high ethanol concentrations and are strong competitors inits respective microbial community. The microbes may be mesophiles orthermophiles. Thermophiles are organisms that grow best at temperaturesabove about 45° C., and are found in all three domains of life:Bacteria, Archaea and Eukarya. Mesophiles generally are active betweenabout 20° C. and 45° C. In an embodiment using a strain of Saccharomycescerevisiae, the strain can come from a commercially available sourcesuch as Biosaf from Lesaffre, Ethanol Red from Phibro, and Lallamandactivated liquid yeast. If the microbe is obtained from a commercialsource, the microbe can be added according to the recommended rate ofthe provider, which is typically based on the expected sugar content perwet ton, where water is included in the mass calculation. The term “wetton” refers at least to the mass unit including water. The recommendedamount can be adjusted according to reaction conditions. The microbeadded can comprise one strain or multiple strains of a particularmicrobe. In one embodiment, the microbes are added at a rate of up to500 mL per wet ton of solid biomass. In a particular embodiment usingcommercially available yeast, about 300 mL of Lallamand yeastpreparation is added per wet ton of solid biomass. In anotherembodiment, an additional yeast strain can be added. For example,Ethanol Red can be added at a rate between about 0.001 kg/wet ton toabout 0.5 kg/wet ton, particularly about 0.1 kg/wet ton. In yet anotherembodiment, another yeast strain can be added, e.g., Biosaf, at a ratebetween about 0.001 kg/wet tone to about 0.5 kg/wet ton, particularlyabout 0.1 kg/wet ton. It is understood that other amounts of any yeaststrain can be added. For example, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1.5times, about 2 times, about 2.5 times, or about 3 times of the providedamounts of microbes can be added.

In certain embodiments, an enzyme is further added. The enzyme can beone that assists in the generation of fermentable sugars from plantmaterials that are more difficult for the microbe to metabolize, such asdifferent cellulosic materials, and/or to improve the value of aneventual by-product serving as animal feed, such as by making the feedmore digestable. The enzyme can also be an antibiotic, such as alysozyme as discussed further below. The enzyme added can include onetype of enzyme or many types of enzymes. The enzyme can come fromcommercially available enzyme preparations. Non-limiting examples ofenzymes that assist in converting certain difficult to metabolize plantmaterials into fermentable sugars include cellulases, hemicellulases,ferulic acid esterases, and/or proteases. Additional examples alsoinclude other enzymes that either provide or assist the provision forthe production of fermentable sugars from the feedstock, or increase thevalue of the eventual feed by-product.

In certain embodiments, the enzymes that assist in converting certaindifficult to metabolize plant materials into fermentable sugars can beproduced by the plant itself, e.g. in-plantae. Examples of plants thatcan produce cellulases, hemicellulases, and other plant-polymerdegrading enzymes may be produced within the growing plants aredescribed in the patent publications and patent WO2011057159,WO2007100897, WO9811235, and U.S. Pat. No. 6,818,803, which show thatenzymes for depolymerizing plant cell walls may be produced in plants.In another embodiment, ensilagement can be used to activate such plantproduced enzymes as well as temper the biomass for further processing.One example is described in patent publication WO201096510. If used,such transgenic plants can be included in the harvest in any amount. Forexample, certain embodiments may employ in-plantae enzymes produced inplants by using particular transgenic plants exclusively as a feedstock,or incorporating the transgenic plants in an interspersed manner withinlike or different crops.

In certain embodiments that include such plant-polymer degradingenzymes, ethanol can be produced from cellulosic fractions of the plant.In a particular embodiment, when Novazymes CTEC2 enzyme was added to asorghum storage system in excess of the recommended amount, about 100times more than the recommended amount, about 152% of the theoreticalethanol conversion efficiency based on the initial free sugar contentwas achieved. While such an amount of enzymes can be added usingcommercially available formulations, doing so can be costly. On theother hand, such an amount of enzymes can be obtained in a more costeffective manner by growing transgenic plants that produce these enzymesat least interspersingly among the biomass crop.

The ethanol production from cellulose occurred during the storage phase,e.g., in silage and was stable for about 102 days of storage, afterwhich the experiment was terminated. This demonstrates that, under theconditions of that particular experiment, an excess of such enzymeactivity results in at least about 52% production of ethanol usingfermentable sugars from cellulose. Not intended to be bound by theory,for certain embodiments, the immediate addition of acid during harvestin the experiment may have lowered the pH, thereby potentially inducingthe enzyme activity, which otherwise could damage the plants if producedwhile the plants were still growing.

In a preferred embodiment, if an enzyme is added, the enzyme can be anyfamily of cellulase preparations. In one embodiment, the cellulosepreparation used is Novozymes Cellic CTec 2 or CTec 3. In anotherembodiment, a fibrolytic enzyme preparation is used, particularly,Liquicell 2500. If used, the amount of enzyme added to degrade plantpolymer can be any amount that achieves the desired conversion of plantmaterial to fermentable sugar, such as the recommended amount. In aparticular embodiment, about 80,000 FPU to about 90,000,000 FPU,preferably about 400,000 FPU to about 45,000,000 FPU, more preferablyabout 800,000 FPU to about 10,000,000 FPU of enzyme is added per wet tonof biomass. The term “FPU” refers to Filter Paper Unit, which refers atleast to the amount of enzyme required to liberate 2 mg of reducingsugar (e.g., glucose) from a 50 mg piece of Whatman No. 1 filter paperin 1 hour at 50° C. at approximately pH 4.8.

In certain other embodiments, selected additive(s) added can includeother substances capable of slowing or controlling bacterial growth.Non-limiting examples of these other substances include antibiotics(including antibiotic enzymes), such as Lysovin (lysozyme) and Lactrol®(Virginiamycin, a bacterial inhibitor). Control of bacterial growth canallow the appropriate microbe to expedite and/or provide the productionof volatile organic compounds. Antibiotic is a general term forsomething which suppresses or kills life. An example of an antibiotic isa bacterial inhibitor. In one embodiment, a selective antibiotic that isintended to impact bacteria and not other microbes is used. One exampleof a selective antibiotic is Lactrol, which affects bacteria but doesnot affect yeasts.

In a particular embodiment, if used, Lactrol can be added at rates ofabout 1 to 20 part-per-million (ppm) w/v (weight Lactrol per volumeliquid) as dissolved in the water phase of the prepared biomassmaterial, for example at about at about 5 ppm w/v. In an embodimentusing an enzyme to control bacterial growth, lysozyme is preferablyused. The lysozyme can come from a commercial source. An exemplarycommercially available lysozyme preparation is Lysovin, which is apreparation of the enzyme lysozyme that has been declared permissiblefor use in food, such as wine.

The enzyme and/or other antibiotic material, if used, can be addedindependently or in conjunction with one another and/or with themicrobe. In certain embodiments, other compounds serving as nutrients tothe microbes facilitating and/or providing the volatile organic compoundproduction can also be added as an additive. The following Table 4provides non-limiting examples of other substances, includingantibiotics, which can be added to the solid biomass.

TABLE 4 Potassium Potassium FermaSure ® (from Lysovin MetabisulfiteBicarbonate Dupont ™) - oxychlorine products including chlorite ThiaminMagnesium Calcium Diammonium Sulfate Pantothenate Phosphate AmmoniaAntibiotics Lactrol Biotin

Yeasts and other microbes that are attached to solids individually, assmall aggregates, or biofilms have been shown to have increasedtolerance to inhibitory compounds. Not intended to be bound by theory,part of the long-term fermentation may be possible or enhanced by suchmicrobial-to-solids binding. As such, the prepared biomass material thatincludes the microbe optimized for microbial binding as well asadditives that may bind microorganisms can experience a greater extentof fermentation and or efficiency of fermentation. Substances providingand/or facilitating long term fermentation is different from substancesthat increase the rate of fermentation. In certain embodiments, anincrease in the rate of fermentation is not as an important factor asthe long-term fermentation, particularly over a period of many weeks ormonths.

The following provides particular amounts of additives applied to onespecific embodiment. If used, the rate and amount of adding an acidvaries with the buffering capacity of the particular solid biomass towhich the particular acid is added. In a particular embodiment usingsulfuric acid, 9.3% w/w sulfuric acid is added at rates of up to about10 liter/ton wet biomass, for example at about 3.8 liter/ton wet biomassto achieve a pH of about 4.2. In other embodiments, the rate will varydepending on the concentration and type of acid, liquid and othercontent and buffering capacity of the particular solid biomass, and/ordesired pH. In this particular embodiment, Lactrol is added at a rate ofabout 3.2 g/wet ton of solid biomass. Yeasts or other microbes are addedaccording to the recommended rate from the provider, such as accordingto the expected sugar content per wet ton. In one particular embodiment,Lallemand stabilized liquid yeast is added at about 18 fl oz per wetton, and Novozymes Cellic CTec2 is added at about 20 fl oz per wet ton.

In a preferred embodiment, selected additive(s) are added to the solidbiomass stream during harvest according to aspects of the inventiondescribed above to generate the prepared biomass material. Preferably,the prepared biomass material is transported to a storage facility toallow for conversion of carbohydrates of the prepared biomass materialinto volatile organic compounds of the desired amount and/or awaitrecovery of the volatile organic compounds. Any suitable transportationmethod and/or device can be used, such as vehicles, trains, etc, and anysuitable method to place the prepared biomass material onto thetransportation means. Non-limiting examples of vehicles that can be usedto transport the biomass material include end-unloading dump trucks,side-unloading dump trucks, and self-unloading silage trucks. In apreferred embodiment, a silage truck is used. In embodiments using aforage harvester to collect the biomass, transportation of such solidbiomass is more efficient than transportation of materials collected byconventional means, such as sugar cane billets, because the bulk densityis higher in the solid biomass cut with a forage harvester. That is,materials chopped into smaller pieces pack more densely than materialsin billets. In one embodiment, the range of bulk densities in a silagetruck varies between about 150 kg/m³ and about 350 kg/m³, for exampleabout 256 kg/m³. Because in certain embodiments, all selected additivesare added during harvest, preferably on the harvester, the microbe maybegin to interact with the biomass during transportation, and in thisway transportation is not detrimental to the overall process.

The biomass, whether prepared or not, is delivered to at least onestorage area or facility. The storage facility can be located anydistance from the harvest site. Selected additive(s) can be added ifthey have not been added already or if additional amounts or types needto be further added to generate the prepared biomass material. In apreferred embodiment, the prepared biomass is stored in at least onepile on a prepared surface for a period of time. The facility canincorporate man-made or natural topography. Man-made structures caninclude existing structures at the site not initially designated forsilage, such as canals and water treatment ponds. Non-limiting examplesof a prepared surface includes a concrete, asphalt, fly ash, or soilsurface. The at least one pile can have any dimension or shape, whichcan depend on operating conditions, such as space available, amount ofbiomass, desired storage duration, etc.

The conversion process of fermentable sugars is an exothermic reaction.Too much heat, however, can be detrimental to the conversion process ifthe temperature is in the lethal range for the microbes in the preparedbiomass material. However, in an embodiment using about 700 wet tons ofbiomass and piling up to about 12 feet, ethanol production and stabilitywere satisfactory. Therefore larger piles will likely not suffer fromoverheating. In one embodiment, an inner portion of the pile maintains atemperature in a range of about 20° C. to about 60° C. for microbes ofall types, including thermophiles. In an embodiment not employingthermophiles, an inner portion of the pile maintains a temperature in arange of about 35° C. to about 45° C.

The prepared biomass material that is stored as at least one pile at thestorage facility can also be referred to as a wet stored biomassaggregate. After addition of the selected additive(s), at least aportion of the solid biomass is converted to volatile organic compounds,such as fermentation of sugars into ethanol. In one embodiment, theprepared biomass material is stored for a period of time sufficient toachieve an anaerobiasis environment. In a preferred embodiment, theanaerobiasis environment is achieved in about 24 hours. In anotherembodiment, the anaerobiasis environment is achieved in more than about4 hours. In yet another embodiment, the anaerobiasis environment isachieved in up to about 72 hours.

The pile can be free standing or formed in another structure, such as asilage bunker, designed to accept silage, including provisions tocollect aqueous runoff and leachate, placement of a tarp over thebiomass, and to facilitate both efficient initial silage truck unloadinginto the bunker as well as removal of the biomass year around. Theindividual bunkers may be sized at about the size to support annualfeedstock requirements of about 700 wet tons to 10,000,000 wet tons ormore. For example, the storage facility may have 50 bunkers, where eachindividual bunker can accept 100,000 wet tons of prepared biomassmaterial for a total of a maximum of about 5 million wet tons of storedmaterial at any one time. In a preferred embodiment where ethanol is thevolatile organic compound of choice, about 14 gallons to about 16gallons of ethanol is recovered per one wet ton of prepared biomassmaterial. The provided numbers are exemplary and not intended to limitthe amount of prepared biomass material a storage facility canaccommodate.

In a particular embodiment, the storage pile further includes a leachatecollection system. In one embodiment, the collection system is used toremove leachate collected from the storage pile. For example, theleachate collection system can be adapted to remove liquid from the pileat certain points during the storage period. In another embodiment, theleachate collection system is adapted to circulate the liquid in thestorage pile. For example, circulation can involve taking at least aportion of the recovered liquid and routing it back to the pile,preferably at or near the top portion. Such recirculation allows forlonger retention time of certain portions of the liquids in the pile,even as the recovery phase of the prepared biomass material begins andportions of the non-liquid component of the prepared biomass materialare sent to the recovery unit. The longer retention time results inlonger microbial reaction time, and hence, higher concentrations oforganic volatile compounds, such as ethanol.

Any suitable leachate collection system known to those skilled in theart can be employed as described. In a particular embodiment, theleachate collection system comprises at least one trough along thebottom of the pile, preferably positioned near the middle, of thestorage pile or bunker if one is used, where the storage pile isprepared at a grade designed to direct liquid from the prepared biomassmaterial to the trough and out to a desired collection receptacle orrouted to other applications.

In another embodiment, the leachate collection system comprises one ormore perforated conduits, preferably pipes made of polyvinyl chloride(PVC), that run along the bottom of the pile to allow the liquidcollected in the conduits to be directed away from the pile.

In one embodiment, as the prepared biomass material is added to thebunker or laid on top of the prepared surface, a tractor or other heavyimplement is driven over the pile repeatedly to facilitate packing. Inone embodiment, the packing ranges from about 7 lbs/ft³ to about 50lbs/ft³ per cubic foot for the prepared biomass material. In a preferredembodiment, the packing is from about 30 lbs/ft³ to about 50 lbs/ft³,particularly about 44 lbs/ft³. In one embodiment, the compacting of theprepared biomass material in a pile facilitates and/or allows ananaerobiasis environment to be achieved in the preferred time periodsdescribed above. In another embodiment, after the packing is performedor during the time the packing is being performed, an air impermeablemembrane is placed on the pile, typically a fit for purpose plastictarp. In a particular embodiment, the tarp is placed on the pile as soonas is practical. For instance, the tar is placed on the pile within a24-hour period.

In one embodiment, the prepared biomass material is stored for at leastabout 24 hours and preferably at least about 72 hours (or 3 days) toallow for production of volatile organic compounds, such as ethanol. Inone embodiment, the prepared biomass material is stored for about threedays, preferably ten days, more preferably greater than ten days. In oneembodiment, the time period for storage of the prepared biomass is about1 day to about 700 days, preferably about 10 to 700 days. In anotherembodiment, the biomass material is stored for up to about three years.In one embodiment, the prepared biomass material is stored for a timeperiod sufficient to allow a conversion efficiency of sugar to at leastone volatile organic compound of at least about 95% of the theoreticalproduction efficiency as calculated through a stoichiometric assessmentof the relevant biochemical pathway. In another embodiment, the preparedbiomass material is stored for a time period sufficient to allow acalculated conversion efficiency of sugar to at least one volatileorganic compound of at least about 100%. In yet another embodiment, theprepared biomass material is prepared with certain additives, such asenzymes, that allow a calculated conversion efficiency of sugar to atleast one volatile organic compound of up to about 150% of thetheoretical value based on the initial amount of available fermentablesugars. Not intended to be bound by theory, it is believed that, at orabove 100% efficiency, the volatile organic compound(s) are producedfrom both the initially available fermentable sugars and fermentablesugars from cellulosic or other polymeric material in the preparedbiomass material, which can be achieved by enzymatic hydrolysis or acidhydrolysis facilitated by certain additive(s) applied to the biomass.

The produced volatile organic products, such as ethanol, remain stablein the stored prepared biomass material for the duration of the storageperiod. In particular, the prepared biomass material can be stored up to700 days without significant degradation to the volatile organiccompounds. “Significant” in this context refers at least to within themargin of error when measuring the amount or concentration of thevolatile organic compounds in the prepared biomass material. In oneembodiment, the margin of error is 0.5%. It has been demonstrated thatethanol remains stable in the pile after at least about 330 days with nosignificant ethanol losses observed. This aspect of embodiments of thepresent invention is important because it provides for at least eightmonths of stable storage, which enables year-round VOCs production andrecovery with a harvest window of only about four months. Embodiments ofthe invention provide significant advantages over the conventionaljust-in-time processing that would only be able to operate during thefour months harvest window per year. That is, embodiments of theinvention allow a plant to operate year-round using only a four-monthharvest window, thereby reducing capitals cost for a plant of the samesize as one used for just-in-time processing.

Also, in an embodiment employing a tarp, it is envisioned that placingsoil or other medium around and on the tarp edges to 1) provide weightfor holding the tarp down; and also 2) to act as a biofilter of theoff-gas from the pile. In such an embodiment, biofilters are efficientfor organics and carbon monoxide detoxification/degradation. Theprepared biomass material can also be stored as compressed modules,drive over piles, bunkers, silos, bags, tubes, or wrapped bales or otheranaerobic storage system.

In one embodiment, the off-gas stream from a pile of prepared biomassmaterial was monitored, and it was found that only small levels oforganics, and also very low levels of nitrogen oxides, were present. Forexample, Tables 5.1, 5.2, and 5.3 below show the analysis of variousoff-gas samples collected during the storage phase of one implementationof certain embodiments of the invention. The designation “BDL” refers toan amount below detectable limit. Summa and Tedlar refer to gas samplingcontainers commercially available.

TABLE 5.1 Con- Con- Nor- tain- tain- mal- er er % % % % % % ized type IDH₂ O₂ N₂ CH₄ CO₂ H₂O CO2 Tedlar A BDL 1.72 7.84 BDL 95.90 5.23 85.21 bagTedlar B BDL 2.30 9.12 BDL 89.97 5.97 82.62 bag Tedlar C BDL 0.71 3.57BDL 97.45 5.54 90.18 bag Tedlar D BDL 0.72 3.18 BDL 97.50 5.97 90.14 bagTedlar E BDL 1.86 7.24 BDL 91.75 7.64 83.26 bag Summa EQ 0.01 5.74 22.140.07 73.74 5.28 66.84 Con- #8 tainer Summa EQ 0.09 3.28 12.89 0.33 84.485.66 78.18 Con- #13 tainer Summa EQ 0.12 3.30 13.01 0.12 84.65 4.9978.70 Con- #16 tainer

TABLE 5.2 Con- Con- tainer tainer % ppmv % ppmv ppmv ppmv ppmv ppmv typeID O₂ CO CO₂ HC NO NO₂ NO_(X) SO₂ Tedlar A 1.6 13 72.7 104 3.8 1.90 5.70BDL bag Tedlar B 4.4 19 66.2 739 2.5 122.90 125.40 6 bag Tedlar C 0.6 2975.3 158 8.9 27.20 36.10 4 bag Tedlar D 0.6 35 75.7 222 7.9 56.50 64.405 bag Tedlar E 4.1 35 66.8 423 3.0 20.30 23.90 4 bag

TABLE 5.3 Con- Con- ppmv tain- tain- 2- ppmv er er ppmv ppmv ppmv pro-ppmv pro- type ID CH2O C2H4O methanol panol ethanol panol Tedlar A 386870 63.4 0.593 78.5 BDL bag Tedlar B BDL 1299 678 0.186 1065 15.2 bagTedlar C 18.2 590 89.2 2.784 171 6.098 bag Tedlar D BDL 941 170 3.031264 7.648 bag Tedlar E BDL 819 389 2.512 634 11.3 bag

Embodiments of the present invention, although relatively uncontained inthe bunker, should be environmentally benign. Even so, certain aspectsof the present invention fit well with using soil or other media as abiofilter placed around and on the bunkers because the escape of gasfrom under the tarp is radial in nature. As such, the vapors have ahigher amount of surface area in contact with the edges of the pile. Inembodiments using a biofilter, vapor phase releases pass through thebiofilter (such as soil or compost) placed near the edge mass beforeentering into the atmosphere. The biofilter retains many potentialenvironmental pollutants and odors released by the storage pile, and iteliminates or greatly reduces the potentially harmful off-gases releasedfrom the storage pile.

In one embodiment, the prepared biomass material is stored until itcontains no more than about 80 wt % liquid. The prepared biomassmaterial is stored until it contains at least about 4 to about 5% higherthan initial content. At this stage, the wet stored biomass aggregate isnot considered “beer” yet since it still contains over about 20% solids.In one embodiment, the prepared biomass material is stored until itcontains between about 2 wt % and about 50 wt % ethanol, and preferablybetween about 4 wt % and about 10 wt % ethanol. The balance of theliquid is primarily water but can contain many other organic compounds,such as acetic acid, lactic acid, etc.

Embodiments of the present invention allow the solid biomass to beharvested in a much shorter harvest window than typical sugar canejuicing operations, which allows for

1) a much larger geographic area where the facilities could be placed,

2) harvest of the crop when the crop has its highest yield potential,

3) harvest of the crop at its highest sugar concentration potential,

4) shorter harvest window still economical, and

5) decoupling the need for taking the juice from the biomass forfermentation.

VOC Recovery

Once the prepared biomass material has been stored for the desiredamount of time and/or contains a desired concentration of volatileorganic compounds, such as ethanol, it can be routed to the VOC recoverysystem for recovery of particular volatile organic compounds. Therecovery system and storage facility can be located any distance fromone another. Embodiments of systems and methods described herein allowflexibility in the geographical location of both and their locationsrelative to each other. In a particular embodiment, the recovery systemis located about 0.5 to about 2 miles from the storage facility. Anysuitable method and/or equipment can be used to transfer the preparedbiomass material from the storage facility to the recovery system. Inone embodiment, a feed hopper is used. In one embodiment, a silagefacer, a front end loader or payloader, a sweep auger or other augersystem can be used to place the prepared biomass material into the feedhopper. The material can be placed directly into the feed hopper or itcan be transferred to by conveyer system, such as belt system. The feedhopper containing the prepared biomass material can then be driven tothe recovery system.

The recovery system is solventless and uses a superheated vapor streamto vaporize the liquid in the prepared biomass material into a gascomponent, which can then be collected. A super-heated vapor is a vaporthat is heated above its saturation temperature at the pressure ofoperation. In a preferred embodiment, after the recovery system reachessteady state, the superheated vapor stream comprises only vaporpreviously evaporated from the prepared biomass material, so that noother gas is introduced, thereby reducing the risk of combustion of thevolatile organic compounds and/or dilution of the recovered productstream of volatile organic compounds. A portion of the vapor is removedas product and the remainder is recycled back for use in transferringheat to fresh incoming prepared biomass material. The remaining solidcomponent is discharged from the system and can have various subsequentuses. The super-heated vapor directly contacts the biomass transferringenergy and vaporizing the liquid present there. The heat or thermalenergy source does not directly contact the prepared biomass material.Thus, the VOC recovery system can also be described as providing“indirect” heat contact.

To provide solventless recovery of volatile organic compounds, therecovery system comprises a compartment that allows superheated vapor toflow in a continuous manner, i.e., as a stream. In one embodiment, thecompartment has a loop shape. In another embodiment, the compartment isa rotating drum. The compartment has an inlet through which the preparedbiomass material can enter. In one embodiment, the inlet comprises apressure tight rotary valve, plug screw, or other similar device, whichcan assist in separating the prepared biomass material to increase thesurface area exposed to the superheated vapor stream.

In yet another embodiment, the system comprises a dewatering mechanismto remove at least a portion of the liquid in the prepared biomassmaterial before the liquid is vaporized. The liquid removal can occurbefore and/or while the prepared biomass material enters thecompartment. The liquid from the prepared biomass material contains atleast one volatile organic compound, which can be recovered by furtherprocessing the liquid, such as feeding the liquid to a distillationcolumn. The liquid can be routed directly to further processing unit,such as a distillation column. Alternatively or in addition to, thesystem further includes a collection unit to collect the liquid removedfrom the prepared biomass material. Any portion of the collected liquidcan then be further processed.

In one embodiment, the dewatering mechanism comprises a componentadapted to squeeze the liquid from the prepared biomass material. Insuch an embodiment, the squeezing can be performed while the preparedbiomass material is being fed into the compartment. For instance, theinlet can comprise a squeezing mechanism to squeeze liquid from theprepared biomass material as it is introduced into the compartment.Alternatively or in addition to, the squeezing can be performedseparately before the prepared biomass material enters the compartment.A non-limiting example of such a squeezing mechanism is a screw plugfeeder.

In one embodiment, the liquid removal mechanism comprises a mechanicalpress. Non-limiting examples of types of mechanical presses include beltfilter presses, V-type presses, ring presses, screw presses and drumpresses. In a particular embodiment of a belt filter press, the preparedbiomass material is sandwiched between two porous belts, which arepassed over and under rollers to squeeze moisture out. In anotherparticular embodiment, a drum press comprises a perforated drum with arevolving press roll inside it that presses material against theperforated drum. In yet another embodiment, in a bowl centrifuge, thematerial enters a conical, spinning bowl in which solids accumulate onthe perimeter.

The compartment provides a space where the superheated vapor stream cancontact the prepared biomass material to vaporize the liquid from theprepared biomass material. The vaporization of at least a portion of theliquid provides a gas component and a solid component of the preparedbiomass material. The system further comprises a separating unit wherethe solid component of the prepared biomass material can be separatedfrom the gas component, so each component can be removed as desired forfurther processing. In one embodiment, the separating unit comprises acentrifugal collector. An example of such centrifugal collector is highefficiency cyclone equipment. In a preferred embodiment, the separatingunit also discharges the solid component from the solventless recoverysystem. There is a separate outlet for the gas component where it canexit the system for further processing, such as distillation. In oneembodiment, the separating unit is further coupled to a second pressuretight rotary valve or the like to extrude or discharge the solidcomponent. In one embodiment, the superheated vapor is maintained at adesired temperature above its saturation temperature by a heat exchangecomponent coupled to a heat source where the superheated vapor does notcontact the heat source. The heat transfer between the heat source andthe system occurs via convection to the superheated vapor. In oneembodiment, the heat source can include electrical elements or hotvapors through an appropriate heat exchanger. In one embodiment, theoperating pressure is in a range from about 1 psig to about 120 psig. Ina preferred embodiment, the operating pressure is in a range from about3 psig to about 40 psig. In a particularly preferred embodiment, thesystem is at an operating pressure of about 60 psig to force the vaporcomponent from the system.

In one embodiment, at start up of the recovery system, the preparedbiomass material is introduced into the compartment via the inlet. Steamis initially used as the superheated vapor to initially vaporize theliquid in the prepared biomass material. The superheated vaporcontinuously moves through the compartment. When the prepared biomassmaterial enters the superheated vapor stream, it becomes fluidized whereit flows through the compartment like a fluid. As the prepared biomassmaterial is introduced, it comes into contact with the superheated vaporstream. Heat from the superheated vapor is transferred to the preparedbiomass material and vaporizes at least a portion of the liquid in theprepared biomass material and is separated from the solid component,which may still contain moisture. The gas component contains volatileorganic compound(s) produced in the prepared biomass material. In apreferred embodiment, as liquid from the prepared biomass materialbegins to vaporize, at least a portion of the vaporized liquid can berecycled in the system as superheated fluid. That is, during any onecycle, at least a portion of the vaporized liquid remains in thecompartment to serve as superheated vapor instead of being collected forfurther processing, until the next cycle where more prepared biomassmaterial is fed into the system.

In a preferred embodiment, during the initial start up procedure, thesuperheated fluid can be purged as needed, preferably continuously(intermittently or constantly), until steady state is achieved where thesuperheated vapor comprises only vaporized liquid of the preparedbiomass material. The gas component and solid component can be collectedvia the respective outlet. Heat can be added continuously(intermittently or constantly) to the system via the heat exchangercoupled to the heat source to maintain the temperature of thesuperheated vapor, to maintain a desired operating pressure in thesystem, or to maintain a target vaporization rate. Various conditions ofthe system, such as flow rate of the superheated vapor stream, pressure,and temperature, can be adjusted to achieve the desired liquid and/orvolatile organic compounds removal rate.

In one embodiment, the collected gas component is condensed for furtherprocessing, such as being transferred to a purification process toobtain a higher concentration of the volatile organic compound(s) ofchoice. In a preferred embodiment, the collected gas component is feddirectly into a distillation column, which provides savings of energynot used to condense the gas component. In another embodiment, the gascomponent is condensed and fed to the next purification step as liquid.

In one embodiment, before entering the recovery phase, the preparedbiomass material has an initial liquid content of about at least 10 wt %and up to about 80 wt % based on the biomass material. In a particularembodiment, the initial liquid content is at least about 50 wt % basedon the biomass material. In one embodiment, the initial liquid contentcomprises from about 2 to 50 wt %, and preferably from about 4 to 10 wt% ethanol based on the initial liquid content.

In one embodiment, the solid component collected contains from about 5wt % to about 70 wt %, and preferably from about 30 wt % to about 50 wt%, liquid depending on the ethanol removal target. In another component,the collected gas component contains between about 1 wt % and about 50wt % ethanol, preferably between about 4 wt % and about 15 wt % ethanol.In one embodiment, the recovery system recovers from about 50% to about100% of the volatile organic compounds contained in the prepared biomassmaterial. The residence time of the prepared biomass varies based on anumber of factors, including the volatile organic compound removaltarget. In one embodiment, the residence time of the prepared biomassmaterial in the compartment is in a range of about 1 to about 10seconds. In one embodiment, the recovery system can be operated betweenabout 0.06 barg and about 16 barg. The term “barg” refers to bar gaugeas understood by one of ordinary skill in the art, and 1 bar equals to0.1 MegaPascal. In one embodiment, the gas in the recovery system has atemperature in a range of about 100° C. to about 375° C., particularlyfrom about 104° C. to about 372° C., and the solid component exiting thesystem has a temperature of less than about 50° C. The collected solidcomponent can be used in other applications. Non-limiting examplesinclude animal feed, feed for a biomass burner to supply process energyor generate electricity, or further converted to ethanol by means of acellulosic ethanol process (either re-ferment in a silage pile, or feedto a pre-treatment unit for any cellulosic ethanol process) or a feedfor any other bio-fuel process requiring ligno-cellulosic biomass.

The operating conditions of the solventless recovery system include atleast one of temperature, pressure, flow velocity, and residence time.Any one or combination of these conditions can be controlled to achievea target or desired removal target, such as the amount of the initialliquid content removed or the amount of the liquid remaining in theseparated liquid component exiting the recovery system. In oneembodiment, at least one operating condition is controlled to achieveremoval of about 10-90 wt %, preferably about 45-65 wt %, and morepreferably about 50 wt %, of the initial liquid content.

In a preferred embodiment, increasing the temperature of the system atconstant pressure will cause the liquid in the biomass to be vaporizedmore quickly and thus for a given residence time will cause a higherpercentage of the liquid in the biomass to be evaporated. The vapor flowrate exiting the system has to be controlled to match the rate ofvaporization of liquid from the biomass in order to achieve steady stateand can also be used as a mechanism to control the system pressure.Increasing the system pressure will cause more energy to be stored inthe vapor phase in the system which can then be used to aid in furtherprocessing or to help move the vapor to the next downstream processingunit. Increasing the biomass residence time in the system causes moreheat to be transferred from the vapor phase to the biomass resulting inmore liquid being vaporized.

In a specific exemplary embodiment, the recovery system comprises aclosed loop pneumatic superheated steam dryer, which can be obtainedfrom commercially available sources. In one embodiment, the closed looppneumatic superheated steam dryer is an SSD™ model of GEA Barr-RosinInc. Other suitable commercially available equipment include theSuperheated Steam Processor, SSP™ from GEA Barr-Rosin Inc, the RingDryer from several companies including GEA Ban-Rosin Inc. and Dupps; theAirless Dryer from Dupps; the QuadPass™ Rotary Drum Dryer fromDuppsEvactherm™, Vacuum Superheated Steam Drying from Eirich; the rotarydrum dryer using superheated vapor from Swiss Combi Ecodry; and theairless dryer from Ceramic Drying Systems Ltd.

Still other types of indirect dryers that could serve as the volatileorganics recovery unit for this process are batch tray dryers,indirect-contact rotary dryers, rotating batch vacuum dryers, andagitated dryers. The basic principle for these dryers is that they willbe enclosed and attached to a vacuum system to remove vapors from thesolids as they are generated (also by lowering the pressure with thevacuum the volatiles are removed more easily). The wet solids contact ahot surface such as trays or paddles, the heat is transferred to the wetsolids causing the liquids to evaporate so they can be collected in thevacuum system and condensed.

FIG. 1 illustrates an exemplary VOC recovery system and processemploying a superheated steam dryer, referenced as system 100. In aparticular embodiment, the superheated steam dry can be obtained fromGEA Ban-Rosin Inc. In FIG. 1, prepared biomass material 1 containingethanol and/or other VOCs following solid state fermentation in thesilage piles is fed into compartment 3 through input 2. In theparticular embodiment shown, input 2 comprises a screw extruder. Asshown in FIG. 1, at least a portion of the liquid of the preparedbiomass material 1 is removed prior to entering compartment 3. Thedewatering mechanism can be a screw plug feeder through which theprepared biomass material 1 passes. At least a portion of the liquidremoved from biomass material 1 can be routed directly to distillationstep 11 via stream 15 without going through recovery system 100.Optionally, a delumper can be coupled to the output of the dewateringmechanism can be used to facilitate introduction of the dewateredbiomass material into compartment 3.

Referring to FIG. 1, recovery system 100 comprises compartment 3, whichcan be pressurized, shown as a conduit that has an appropriate diameter,length and shape, adapted to provide the desired operating conditions,such as residence time of prepared biomass material 1, heat transfer tothe superheated vapor, and operating pressure and temperature. Afterentering compartment 3, during steady state operation, prepared biomassmaterial 1 contacts superheated vapor flowing through system 100 at adesired temperature and becomes fluidized. As described above, in apreferred embodiment, the superheated vapor, or at least a portionthereof, is vapor component obtained from prepared biomass materialspreviously fed into system 100 for VOC recovery. The fluidized biomassflows through compartment 3 at a target flow rate and remains in contactwith the superheated vapor for a target residence time sufficient toevaporate the desired amount of liquid from prepared biomass material 1.In the embodiment shown, the flow of the superheated vapor and preparedbiomass material 1 through system 100 is facilitated by system fan 14.System 100 can have one or more fans. The flow rate or velocity of thesuperheated vapor and biomass material 1 can be controlled by system fan14. Biomass material 1 flows through compartment 3 and reachesseparating unit 4, which is preferably a cyclone separator, where avapor component and a solid component of biomass material 1 areseparated from each other. As shown, the vapor component is routed awayfrom the solid component via overhead stream 5 and the remaining portionof biomass material 1 is considered a solid component, which isdischarged from separating unit 4 as solid component 7, preferably byscrew extruder 6. At least a portion of the discharged solid component 7can be used as animal feed, burner fuel, or biomass feedstock for otherbio-fuels processes. For example, at least a portion of solid component7 can serve as feedstock for process 400 that further processeslignocellulosic material contained in solid component 7. Process 400 isillustrated in FIG. 3 and correspondingly further discussed below.Referring to FIG. 1, a portion of the vapor component, referenced asstream 8, is retained and recycled as a portion of the superheated vaporused to vaporize newly introduced prepared biomass material. In theembodiment shown, the retained vapor component in stream 8 is routedthrough heat exchanger 9 to heat it to the target operating temperature.The heat source can include steam, electricity, hot flue gases or anyother applicable heating source known to those skilled in the art.

In a preferred embodiment, the temperature is controlled such that thepressure in the system is maintained at the target and there is adequateenergy present to evaporate the desired amount of liquid. The pressurecan also be controlled by the flow rate of the superheated vapor streamand the heat input to heat exchanger 9. Preferably, recovery system 100operates continuously where prepared biomass material 1 is continuouslyfed at a desired rate, and vapor component 10 and solid component 6 arecontinuously removed at a continuous rate. In a preferred embodiment,“fresh” vapor component 8 from one run is retained continuously at atarget rate to be used as the superheated vapor stream for the next run.Any of these rates are adjustable to achieve the desired operatingconditions. As mentioned, system fan 14 circulates the superheated vaporstream through system 100 and can be adjusted to obtain the target flowrate or velocity.

Referring to FIG. 1, the remaining portion of vapor component stream 5,represented as numeral 10 is routed to a distillation step 11. Dependingon the distillation configuration, vapor component portion 10 may becondensed before further purification or preferably fed directly intothe distillation column as a vapor. In a preferred embodiment, thedistillation product from distillation step 11 has an ethanol content ofabout 95.6 wt % ethanol (the ethanol/water azeotrope), which can furtherbe purified to above about 99 wt % using common ethanol dehydrationtechnology, which is shown as step 12. The final ethanol product 13 willthen typically be used as a biofuel for blending with gasoline.

FIG. 2 illustrates another exemplary recovery system and processemploying a superheated steam dryer, referenced as system 200 that isrepresentative of the Ring Dryer provided by various manufacturers.Prepared biomass material 201 is fed into system 200 through input 202,which preferably comprises a screw extruder. In one embodiment, least aportion of the liquid of the prepared biomass material 201 is removedprior to entering system 200. The dewatering mechanism can be a screwplug feeder through which the prepared biomass material 201 passes. Atleast a portion of the liquid removed from biomass material 201 can berouted directly to distillation step 211 via stream 215 without goingthrough recovery system 200. Optionally, a delumper can be coupled tothe output of the dewatering mechanism can be used to facilitateintroduction of the dewatered biomass material into compartment 203.

Referring to FIG. 2, recovery system 200 comprises compartment 203,which preferably comprises a rotating drum that provides the targetoperating conditions for VOC recovery, including residence time ofprepared biomass material 201, heat transfer to the superheated vapor,and operating pressure and temperature. After entering compartment 203,during steady state operation, prepared biomass material 201 contactssuperheated vapor flowing through system 200 at the operatingtemperature and flow rate and becomes fluidized. As described above, ina preferred embodiment, the superheated vapor, or at least a portionthereof, is the vapor component obtained from prepared biomass materialpreviously fed into system 200 for VOC recovery. The fluidized biomassflows through compartment 203 at a target flow rate and remains incontact with the superheated vapor for the target residence time toachieve the target vaporization of liquid from the biomass. Thefluidized biomass then reaches separating unit 204, which is preferablya cyclone separator, where the vapor component and solid component areseparated from each other. As shown, the vapor component is routed awayfrom the solid component through overhead stream 205, and solidcomponent 207 is discharged from separating unit 204. As shown, solidcomponent 207 exits system 100 via extruder 206 and at least a portionof it can serve as feedstock for process 400, which further processeslignocellulosic material contained in solid component 207. Process 400is illustrated in FIG. 3 and correspondingly further discussed below.Solid component 207 can be directly routed to process 400. In additionto or alternatively, solid component 207 can be transported to be fedinto process 400.206 is released from separating unit 204. A portion ofthe vapor component, referenced as stream 208, is retained and recycledas a portion of the superheated vapor used to vaporize newly introducedprepared biomass material. As shown, retained vapor component 208 isrouted through heat exchanger 209 to heat it to the desired temperature.The heat source or thermal energy source can include steam, electricity,hot flue gases or any other desired heating source. As shown, hot fluegas is used. The temperature is controlled such that the pressure in thesystem is maintained at the target and there is adequate energy presentto evaporate the desired amount of liquid. The pressure can also becontrolled by the flow rate of the superheated vapor stream and the heatinput to heat exchanger 209.

Referring to FIG. 2, the remaining portion of vapor component stream205, represented as numeral 210 is routed to a distillation step.Depending on the distillation configuration, vapor component portion 210may be condensed before further purification or preferably fed directlyinto the distillation column as a vapor. The product from thedistillation step can further be concentrated using known processes.

Preferably, recovery system 200 operates continuously where preparedbiomass material 201 is continuously fed at a desired rate, and vaporcomponent 210 and solid component 206 are continuously removed at acontinuous rate. In a preferred embodiment, “fresh” vapor component 208from one run is retained continuously at a target rate to be used as thesuperheated vapor stream for the next run. All these rates areadjustable to achieve the desired operating conditions. System fan 214creates a circulating loop of superheated vapor stream and can beadjusted to obtain the target flow rate.

By using a solventless recovery system according to aspects of thepresent invention, the points of heat transfer in the system, i.e.,addition of heat to the system and heat transfer to the prepared biomassmaterial, take place in the vapor phase in a preferred embodiment, whichprovides an advantage cause vapor phase heat transfer (convection) ismore efficient than solid phase heat transfer (conduction) in theprepared biomass material, which is a bad conductor because it hasinsulating properties. As mentioned above, in certain embodiments, oncesteady state is reached no vapor other than that vaporized from theliquid of the prepared biomass material contacts the solid component andgas component of the prepared biomass material in the system, whichprevents or reduces dilution that would come from the addition ofprocess steam or other vapor to replenish the superheated vapor stream.The collected gas component can be fed directly to a distillation columnfor separation of the desired volatile organic compound(s), which canprovide significant energy savings. The advantage of this system is thatthe vapors that contact the wet solids are only those vapors that havebeen previously removed from the solids so that there is no dilution orexplosion risk, etc.

Further Processing of Lignocellulosic

Referring to FIGS. 1 and 2, at least a portion of the solid component,such as solid components 7 and component 207, discharged from therecovery system, such as systems 100 and 200, can serve as feedstock tofurther processing system 400 and be further processed to generatefermentable sugars. The solid component serving as feedstock to furtherprocessing system 400 may be referred to as “bio-based feedstock,”“solid component feedstock,” or “biomass feedstock.” Further processingsystem 400 treats the lignocellulosic material in the solid component togenerate fermentable sugars that can be used in subsequent reactions,such as additional fermentation. In a preferred embodiment, the furtherprocessing system, such as system 400, is located near the VOC recoverysystem, such as system 100 or 200, and is coupled to the VOC recoverysystem so that at least a portion of the solid component discharged fromthe recovery system is directly routed as feedstock to furtherprocessing system 400, which is preferably operated in a continuous orsemi-continuous flow mode. In that preferred embodiment, the solidcomponent feedstock is in an entrained engineered system where it isalready flowing in an engineered system instead of requiring a mechanismto take it from storage and introduce it to the further processingsystem. Further, embodiments that couple the VOC recovery system to thefurther processing system can allow for production of volatile organiccompounds from various sources, e.g., readily available fermentablesugars and lignocellulosic material, at one site, which reduces storage,handling, and transportation costs associated with other feedstocksources, which are not already in an entrained system. Such embodimentscan also provide a continuous supply of feedstock that is alreadyparticle size reduced in contrast to conventional feedstock that oftenrequires storage, transportation, and/or size reduction at or prior toarriving at the facility for additional processing of lignocellulosicmaterial, which reduces the particular associated costs. Alternativelyor in addition, the solid component can be transported to other furtherprocessing systems located at a different location. The solid componentcan be pelletized or further formatted to facilitate transport and/orreduce transportation costs. In embodiments of the invention, the solidcomponent is already particle size reduced, which reduces the cost anddifficulties of pelletization or other formatting processes as comparedto other feedstock sources.

In certain embodiments, the further processing comprises contacting atleast a portion of the solid component with a solution adapted tofacilitate saccharification. The term “saccharification” has itsordinary meaning, which refers at least to the process of converting acomplex carbohydrate (such as starch or cellulose) into simple orfermentable sugars. Any saccharification process or any combination ofsaccharification process can be used, such as chemical and/or enzymatic.FIG. 3 provides two exemplary saccharification routes forlignocellulosic material: one via concentrated acid hydrolysis and theother via pretreatment and enzymatic hydrolysis. In a preferredembodiment, the saccharification process comprises pretreating the solidcomponent feedstock for subsequent enzymatic hydrolysis. It isunderstood that the pretreatment of the solid component feedstock canalso result in partial or at least some saccharification. Pretreatmentis preferred because the lignocellulose is recalcitrant to enzymatichydrolysis because of its structural complexity. Pretreatment of thesolid component feedstock can improve its enzymatic digestibility,typically by removing hemicellulose and making the cellulose moreaccessible to cellulase enzymes. A variety of chemical and mechanicalpretreatment methods are contemplated, including but not limited to,dilute acid, hot-water, ammonia, alkali, SPORL, steam explosion, ionicliquid, organosolv, etc., which, have been well described in theliterature (see, e.g. Zhu and Pan (2010), Bioresource Technology,101:4992-5002; Hendriks and Zeeman (2009), Bioresource Technology,100:10-18, the disclosures of both articles are herein incorporated byreference in their entireties for all purposes.)

For example, in one embodiment, pretreatment comprises using hot waterin a range from about 170 degrees C. to about 200 degrees C. In anotherembodiment, pretreatment comprises using a high temperature,dilute-sulfuric acid process, which effectively hydrolyzes thehemicellulosic portion of the biomass to soluble sugars and exposes thecellulose so that enzymatic saccharification can be successful. In oneembodiment, the temperature of the pretreatment with the dilute acidsolution is in a range from about 140 degrees C. to about 170 degrees C.The parameters which can be employed to control the conditions of thedilute acid pretreatment include time, temperature, and acid loading.These are often combined in a mathematical equation termed the combinedseverity factor. In general, the higher the acid loading employed, thelower the temperature that can be employed in the pretreatment.Conversely, the lower the temperature used, the longer the pretreatmentprocess takes.

In one embodiment, further processing system 400 further includessubject at least a portion of the pretreated product to enzymatichydrolysis to generate additional fermentable sugars. Additionalinformation regarding enzymatic hydrolysis is further provided below. Ina particular embodiment, the fermentable sugars from further processingof lignocellulosic material can then be fermented using a variety ofmicrobes as described herein. For example, using a microbe adapted toproduce a hydrocarbon. This can generally be referred to aslignocellulosic fermentation.

Referring to FIGS. 1 and 2, in one embodiment, at least a portion ofliquid from the lignocellulosic fermentation, which contains VOCs, canbe routed via stream 430 to join distillation process 11 or 211 of vaporcomponent 10 or 210 and/or liquid product 15 or 215 recovered fromprepared biomass 1 or 201 using solventless recovery system 100 or 200as described above. Likewise, the VOCs in at least a portion of anysolid material from the lignocellulosic fermentation in furtherprocessing 400 can be recovered using the solventless recovery system100 or 200, as indicated by stream 432. Accordingly, certain embodimentsof the invention can provide for an integrated overall system forgeneration of VOCs from readily available fermentable sugars in biomass,recovery of those VOCs, processing lignocellulosic material from thefirst round of fermentation and recovery, generating additional VOCsfrom lignocellulosic material, and recovery of same. Such a system inthose embodiments do not require additional equipment cost, and thuscapital investment, where the same equipment can be used for all VOCsproduction.

In a particularly preferred embodiment, an acid solution comprising atleast one alpha.-hydroxysulfonic acid is used. The α-hydroxysulfonicacid is effective for hydrolyzing the biomass to fermentable sugars likepentose such as xylose at lower temperature, e.g., about 100° C. forα-hydroxymethane sulfonic acid or α-hydroxyethane sulfonic acid,producing little to no furfural in the process. A portion of thecellulose has also been show to hydrolyze under these comparatively mildconditions. It has been found that other polysaccharides such as starchare also readily hydrolyzed to component sugars by α-hydroxy sulfonicacids. Further, the α-hydroxysulfonic acid is reversible to readilyremovable and recyclable materials unlike mineral acids such assulfuric, phosphoric, or hydrochloric acid. The lower temperatures andpressures employed in the biomass treatment leads to lower equipmentcost. Biomass pretreated in this manner has been shown to be highlysusceptible to additional saccharification, especially enzyme mediatedsaccharification.

The alpha-hydroxysulfonic acids of the general formula

where R₁ and R₂ are individually hydrogen or hydrocarbyl with up toabout 9 carbon atoms that may or may not contain oxygen can be used inthe treatment of the instant invention. The alpha-hydroxysulfonic acidcan be a mixture of the aforementioned acids. The acid can generally beprepared by reacting at least one carbonyl compound or precursor ofcarbonyl compound (e.g., trioxane and paraformaldehyde) with sulfurdioxide or precursor of sulfur dioxide (e.g., sulfur and oxidant, orsulfur trioxide and reducing agent) and water according to the followinggeneral equation 1.

where R₁ and R₂ are individually hydrogen or hydrocarbyl with up toabout 9 carbon atoms or a mixture thereof.

Illustrative examples of carbonyl compounds useful to prepare thealpha-hydroxysulfonic acids include

R₁═R₂═H (formaldehyde)

R₁═H, R₂═CH₃ (acetaldehyde)

R₁═H, R₂═CH₂CH₃ (propionaldehyde)

R₁═H, R₂═CH₂CH₂CH₃ (n-butyraldehyde)

R₁═H, R₂═CH(CH₃)₂ (i-butyraldehyde)

R₁═H, R₂═CH₂OH (glycolaldehyde)

R₁═H, R₂═CHOHCH₂OH (glyceraldehdye)

R1=H, R2=C(═O)H (glyoxal)

R₁═R₂═CH₃ (acetone)

R₁═CH₂OH, R₂═CH₃ (acetol)

R₁═CH₃, R₂═CH₂CH₃ (methyl ethyl ketone)

R₁═CH₃, R₂═CHC(CH₃)₂ (mesityl oxide)

R₁═CH₃, R₂═CH₂CH(CH₃)₂ (methyl i-butyl ketone)

R₁, R₂═(CH₂)₅ (cyclohexanone) or

R₁═CH₃, R₂═CH₂Cl (chloroacetone)

The carbonyl compounds and its precursors can be a mixture of compoundsdescribed above. For example, the mixture can be a carbonyl compound ora precursor such as, for example, trioxane which is known to thermallyrevert to formaldehyde at elevated temperatures or an alcohol that maybeconverted to the aldehyde by dehydrogenation of the alcohol to analdehyde by any known methods. An example of such a conversion toaldehyde from alcohol is described below. An example of a source ofcarbonyl compounds maybe a mixture of hydroxyacetaldehyde and otheraldehydes and ketones produced from fast pyrolysis oil such as describedin “Fast Pyrolysis and Bio-oil Upgrading, Biomass-to-Diesel Workshop”,Pacific Northwest National Laboratory, Richland, Washington, Sep. 5-6,2006. The carbonyl compounds and its precursors can also be a mixture ofketones and/or aldehydes with or without alcohols that may be convertedto ketones and/or aldehydes, preferably in the range of 1 to 7 carbonatoms.

The preparation of alpha-hydroxysulfonic acids by the combination of anorganic carbonyl compounds, SO₂ and water is a general reaction and isillustrated in equation 2 for acetone.

The alpha-hydroxysulfonic acids appear to be as strong as, if notstronger than, HCl since an aqueous solution of the adduct has beenreported to react with NaCl freeing the weaker acid, HCl (see U.S. Pat.No. 3,549,319). The reaction in equation 1 is a true equilibrium, whichresults in facile reversibility of the acid. That is, when heated, theequilibrium shifts towards the starting carbonyl, sulfur dioxide, andwater (component form). If the volatile components (e.g. sulfur dioxide)is allowed to depart the reaction mixture via vaporization or othermethods, the acid reaction completely reverses and the solution becomeseffectively neutral. Thus, by increasing the temperature and/or loweringthe pressure, the sulfur dioxide can be driven off and the reactioncompletely reverses due to Le Châtelier's principle, the fate of thecarbonyl compound is dependent upon the nature of the material employed.If the carbonyl is also volatile (e.g. acetaldehyde), this material isalso easily removed in the vapor phase. Carbonyl compounds such asbenzaldehyde, which are sparingly soluble in water, can form a secondorganic phase and be separted by mechanical means. Thus, the carbonylcan be removed by conventional means, e.g., continued application ofheat and/or vacuum, steam and nitrogen stripping, solvent washing,centrifugation, etc. Therefore, the formation of these acids isreversible in that as the temperature is raised, the sulfur dioxideand/or aldehyde and/or ketone can be flashed from the mixture andcondensed or absorbed elsewhere in order to be recycled. It has beenfound that these reversible acids, which are approximately as strong asstrong mineral acids, are effective in biomass treatment reactions. Ithad been found that these treatment reactions produce very few of theundesired byproducts, furfurals, produced by other conventional mineralacids. Additionally, since the acids are effectively removed from thereaction mixture following treatment, neutralization with base and theformation of salts to complicate downstream processing is substantiallyavoided. The ability to reverse and recycle these acids also allows theuse of higher concentrations than would otherwise be economically orenvironmentally practical. As a direct result, the temperature employedin biomass treatment can be reduced to diminish the formation ofbyproducts such as furfural or hydroxymethylfurfural.

In some embodiments, the reactions described are carried out in anysystem of suitable design, including systems comprising continuous-flow(such as CSTR and plug flow reactors), batch, semi-batch or multi-systemvessels and reactors and packed-bed flow-through reactors. For reasonsstrictly of economic viability, it is prefferable that the invention ispracticed using a continuous-flow system at steady-state equilibrium. Inone advantage of the process in contrast with the dilute acidspretreatment reactions where residual acid is left in the reactionmixture (<1% wt. sulfuric acid), the lower temperatures employed usingthese acids (10 to 20% wt.) results in substantially lower pressures inthe reactor resulting in potentially less expensive processing systemssuch as plastic lined reactors, duplex stainless reactors, and 2205 typereactors.

In one embodiment (not shown), at least a portion of product stream oftreated lignocellulosic material can further be subject to enzymatichydrolysis to generate additional fermentable sugars. Additionalinformation regarding enzymatic hydrolysis is further provided below. Ina particular embodiment, the fermentable sugars from further processingof lignocellulosic material can then be fermented using a variety ofmicrobes as described above to generate a plurality of volatile organiccompounds, including hydrocarbon precursor compounds that can beconverted to hydrocarbons. This can generally be referred to aslignocellulosic fermentation.

In some embodiments, a plurality of reactor vessels may be used to carryout the hydrolysis reaction. These vessels may have any design capableof carrying out a hydrolysis reaction. Suitable reactor vessel designscan include, but are not limited to, batch, trickle bed, co-current,counter-current, stirred tank, or fluidized bed reactors. Staging ofreactors can be employed to achieve the optimal or desired economicalsolution. The remaining biomass feedstock solids may then be optionallyseparated from the liquid stream to allow more severe processing of therecalcitrant solids or pass directly within the liquid stream to furtherprocessing that may include enzymatic hydrolysis, fermentation,extraction, distillation and/or hydrogenation. In another embodiment, aseries of reactor vessels may be used with an increasing temperatureprofile so that a desired sugar fraction is extracted in each vessel.The outlet of each vessel can then be cooled prior to combining thestreams, or the streams can be individually fed to the next reaction forconversion.

Suitable reactor designs can include, but are not limited to, abackmixed reactor (e.g., a stirred tank, a bubble column, and/or a jetmixed reactor) may be employed if the viscosity and characteristics ofthe partially digested bio-based feedstock and liquid reaction media issufficient to operate in a regime where bio-based feedstock solids aresuspended in an excess liquid phase (as opposed to a stacked piledigester). It is also conceivable that a trickle bed reactor could beemployed with the biomass present as the stationary phase and a solutionof alpha-hydroxysulfonic acid passing over the material.

The treatment reaction product contains fermentable sugar ormonosaccharides, such as pentose and/or hexose that is suitable forfurther processing.

In one embodiment, the product stream from any pretreatment process canfurther be hydrolyzed by other methods, for example by enzymes tofurther hydrolyze the biomass to sugar products containing pentose andhexose (e.g., glucose) and fermented to produce alcohols such asdisclosed in US Publication No. 2009/0061490 and U.S. Pat. No. 7,781,191which disclosures are hereby incorporated by reference.

The process can be carried out with any type of cellulase enzymes,regardless of their source. Non-limiting examples of cellulases whichmay be used include those obtained from fungi of the genera Aspergillus,Humicola, and Trichoderma, Myceliophthora, Chrysosporium and frombacteria of the genera Bacillus, Thermobifida and Thermotoga. In someembodiments, the filamentous fungal host cell is an Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

The cellulase enzyme dosage is chosen to convert the cellulose of thepretreated feedstock to glucose. For example, an appropriate cellulasedosage can be about 0.1 to about 40.0 Filter Paper Unit(s) (FPU or IU)per gram of cellulose, or any amount there between. The term FilterPaper Unit(s) refers to the amount of enzyme required to liberate 2 mgof reducing sugar (e.g., glucose) from a 50 mg piece of Whatman No. 1filter paper in 1 hour at 50° C. at approximately pH 4.8.

In practice, the hydrolysis may be carried out in a hydrolysis system,which may include a series of hydrolysis reactors. The number ofhydrolysis reactors in the system depends on the cost of the reactors,the volume of the aqueous slurry, and other factors. The enzymatichydrolysis with cellulase enzymes produces an aqueous sugar stream(hydrolyzate) comprising glucose, unconverted cellulose, lignin andother sugar components. The hydrolysis may be carried out in two stages(see U.S. Pat. No. 5,536,325, which is incorporated herein byreference), or may be performed in a single stage.

In one embodiment, the treated solid component comprising fermentablesugars can then be fermented by one or more microorganism to produce afermentation broth comprising the desired chemical. In thelignocellulosic fermentation system, any one of a number of knownmicroorganisms may be used to convert sugar to the desired fermentationproducts. The microorganisms convert sugars, including, but not limitedto glucose, mannose and galactose present in the treated solid componentor hydrolysate to a fermentation product. A particular fermentationproduct is a hydrocarbon. However, other compounds can be generated byadding the appropriate organism.

In one embodiment, the lignocellulosic fermentation comprises using amicroorganism adapted to produce a hydrocarbon. An example of such amicroorganism is disclosed in PCT Application No. PCT/EP2013/053600, thedisclosure of which is incorporated herein by reference.

In one embodiment, a recombinant host cell, such as a recombinantmicro-organism is adapted to express at least one of the followingenzymes: a fatty acid reductase (LuxC), a fatty acyl transferase (LuxD),a fatty aldehyde synthetase (LuxE), and an aldehyde decarbonylase.Coexpression of the fatty acid reductase, fatty aldehyde synthetase,fatty acyl transferase, and aldehyde decarbonylase enzymes can becollectively referred to as CEDDEC. At least one fatty acid reductase,at least one fatty aldehyde synthetase, and at least one fatty acyltransferase forms a fatty acid reductase complex. Contact of a fattyacid substrate with a fatty aldehyde synthetase forms a fatty acidaldehyde. Contact of the fatty acid aldehyde with at least one aldehydedecarbonylase forms a hydrocarbon. The host cell may be recombinant andmay, for example, be a genetically modified microorganism to express atleast one, and preferably all, of these enzymes: a fatty acid reductase,fatty aldehyde synthetase, a fatty acyl transferase, and an aldehydedecarbonylase. These enzymes may each be expressed by a recombinant hostcell, either within the same host cell or in separate host cells. Thehydrocarbon may be secreted from the host cell in which it is formed.Hydrocarbons produced can include alkanes and alkenes of the appropriatechain length for diesel or aviation fuel, namely tridecane, pentadecane,pentadecene, hexadecene, heptadecane, and heptadecene.

In one embodiment, at least some of the fatty acid substrate isobtainable by contacting a fatty acyl-ACP with at least one acyl-ACPthioesterase. The term acyl-ACP thioesterase is an enzyme in the classEC 3.1.2.14, capable of catalysing the release of free fatty acid fromfatty acyl-ACP. The acyl-ACP thioesterase may be, for example, apolypeptide having at least 50% sequence identity to SEQ ID NO:5(thioesterase protein from Cinnamomum camphora).

In one embodiment, at least some of the fatty acyl-ACP is obtainable bycontacting a keto acyl CoA and a malonyl-ACP with at least one3-ketoacyl-ACP synthase III (KASIII). This is an enzyme in class EC2.3.1.180, capable of catalysing the reaction of a keto acyl CoA and amalonyl-ACP to form fatty acyl-ACP. The 3-ketoacyl-ACP synthase III maybe a polypeptide having at least 50% sequence identity to SEQ ID NO:6(Bacillus subtilis enzyme KASIII).

In this embodiment, at least some of the keto acyl-CoA may be obtainableby contacting a keto acid with a branched-chain ketodehydrogenasecomplex. This is an enzyme or complex of enzymes capable of catalysingthe conversion of a keto acid to a keto acyl-CoA. For example, thebranched-chain ketodehydrogenase complex may comprise a polypeptide inclass EC 1.2.4.4 (for example having at least 50% sequence identity toSEQ ID NO:7; B. subtilis BCKD subunit E1α) and a further polypeptide inclass EC 1.2.4.4 (for example having at least 50% sequence identity toSEQ ID NO:8; B. subtilis BCKD subunit E1β) and a polypeptide in class EC2.3.1.168 (for example having at least 50% sequence identity to SEQ IDNO:9; B. subtilis BCKD subunit E2) and a polypeptide in class EC 1.8.1.4(for example having at least 50% sequence identity to SEQ ID NO:10; B.subtilis BCKD subunit E3). In an embodiment, the branched-chainketodehydrogenase complex is a single polypeptide comprising all of theamino acid sequences SEQ ID NOs:7-10.

These other enzymes described herein (i.e., an acyl-ACP thioesteraseand/or a 3-ketoacyl-ACP synthase III and/or a branched-chainketodehydrogenase complex) may also be expressed by a micro-organism.Preferably, the enzymes are exogenous, i.e., not present in the cellprior to modification, having been introduced using microbiologicalmethods such as are described herein. Furthermore, the enzymes may eachbe expressed by a recombinant host cell, either within the same hostcell or in separate host cells. The hydrocarbon may be secreted from thehost cell in which it is formed.

The host cell may be genetically modified by any manner known to besuitable for this purpose by the person skilled in the art. Thisincludes the introduction of the genes of interest on a plasmid orcosmid or other expression vector which may be capable of reproducingwithin the host cell. Alternatively, the plasmid or cosmid DNA or partof the plasmid or cosmid DNA or a linear DNA sequence may integrate intothe host genome, for example by homologous recombination. To carry outgenetic modification, DNA can be introduced or transformed into cells bynatural uptake or mediated by well-known processes such aselectroporation. Genetic modification can involve expression of a geneunder control of an introduced promoter. The introduced DNA may encode aprotein which could act as an enzyme or could regulate the expression offurther genes.

In one embodiment, such a host cell may comprise a nucleic acid sequenceencoding a fatty acid reductase and/or a fatty aldehyde synthetaseand/or a fatty acyl transferase and/or an aldehyde decarbonylase and/oran acyl-ACP thioesterase and/or a 3-ketoacyl-ACP synthase III and/or abranched-chain ketodehydrogenase complex. The nucleic acid sequencesencoding the enzymes may be exogenous, i.e., not naturally occurring inthe host cell.

Therefore, in one embodiment, there is a recombinant host cell, such asa micro-organism, comprising at least one polypeptide which is a fattyacid reductase in class EC 1.2.1.50, for example, having an amino acidsequence at least 50% identical to SEQ ID NO:1 (e.g., SEQ ID NO:1, 28 or29), and comprising at least one polypeptide which is a fatty aldehydesynthetase in class EC 6.2.1.19, for example, having an amino acidsequence at least 50% identical to SEQ ID NO:2 (e.g., SEQ ID NO:2, 32 or33), and comprising at least one polypeptide which is a fatty acyltransferase in class EC 2.3.1.-, for example, having an amino acidsequence at least 50% identical to SEQ ID NO:3 (e.g., SEQ ID NO:3, 30 or31). The cell may also comprise at least one polypeptide which is analdehyde decarbonylase in class EC 4.1.99.5, for example, having anamino acid sequence at least 50% identical to SEQ ID NO:4, or afunctional variant or fragment of any of these sequences. Therecombinant host cell may comprise a polypeptide comprising all of SEQID NOs:1-4 and/or amino acid sequences at least 50% identical to all ofSEQ ID NOs:1-3 (e.g., amino acid sequences selected from SEQ IDNOs:28-33, as outlined above) and at least 50% identical to SEQ ID NO:4.The recombinant host cell may comprise the polynucleotide sequences SEQID NOs:11-14 and/or the sequences SEQ ID NOs:13 & 15 and/or thesequences SEQ ID NOs:13-16 and/or any combination of these specificcombinations.

The recombinant host cell may further comprise: at least one acyl-ACPthioesterase in class EC 3.1.2.14 (e.g., having an amino acid sequencewhich is at least 50% identical to any of SEQ ID NOs:5 or a functionalvariant or fragment thereof); and/or at least one 3-ketoacyl-ACPsynthase III in class EC 2.3.1.180 (e.g., having an amino acid sequencewhich is at least 50% identical to any of SEQ ID NOs:6 or a functionalvariant or fragment thereof); and/or at least one branched-chainketodehydrogenase complex comprising enzymes in classes EC 1.2.4.4,2.3.1.168 and 1.8.1.4 (e.g., comprising one or more amino acidsequence(s) each being at least 50% identical to any of SEQ ID NOs:7-10or a functional variant or fragment thereof); and/or at least onepolynucleotide encoding at least one of these enzymes and/or functionalfragments or variants of these. The cell may also be modified to produceincreased levels of fatty acid which may be used by the fatty acidreductase and fatty aldehyde synthetase and fatty acyl transferase as asubstrate to form a fatty aldehyde which may then be converted to ahydrocarbon by the decarbonylase. The recombinant host cell may alsocomprise one or more transport proteins for transporting hydrocarbon(s)out of the cell.

FIG. 4 is a schematic detailing the genetic elements (solid lines)introduced into E. coli cells to produce bespoke alkanes, theirrelationship with the endogenous genes (dashed lines) and the de novometabolic pathway (the boxes represent genes whilst circles representmetabolic intermediates. Key to metabolites: ILV, isoleucine, leucineand valine; MDHLA, methyl-butan/propanoyl-dihydrolipoamide-E. Key togenes: ilvE, endogenous branched chain amino acid aminotransferase; E1and E1β, branched chain alpha keto acid decarboxylase/dehydrogenase E1αand β subunits from B. subtilis; E2, dihydrolipoyl transacylase from B.subtilis; E3, dihydrolipoamide dehydrogenase from B. subtilis (recycleslipoamide-E for use by E1 subunits); KASIII, keto-acyl synthase III(FabH2) from B. subtilis; accA to accD, endogenous acetyl-CoAcarboxylase genes; fabH, endogenous beta-Ketoacyl-ACP synthase III;tesA, endogenous long chain thioesterase; thioesterase, Myristoyl-acylcarrier protein thioesterase from C. camphora; luxD, acyl transferase,from P. luminescens; luxC and luxE, fatty acid reductase andacyl-protein synthetase from P. luminescens; AD, aldehyde decarbonylasefrom N. punctiforme).

PCT/EP2013/053600 has demonstrated certain aspects of the production ofhydrocarbon described herein, including conversion of exogenous fattyacid to alkane via the cyanobacterial alkane biosynthetic pathway,production of alkanes and alkenes via the FAR/NpAD pathway; expressionof the camphor FatB1 thioesterase gene in E. coli increases the poolsize of tetradecanoic acid, production of tridecane in E. coli cells;production of branched fatty acids in E. coli, and production ofbranched pentadecane in E. coli cells.

A suitable polynucleotide may be introduced into the cell by homologousrecombination and/or may form part of an expression vector comprising atleast one of the polynucleotide sequences SEQ ID NOs:11-25 or acomplement thereof. Such an expression vector forms a third aspect ofthe invention. Suitable vectors for construction of such an expressionvector are well known in the art (examples are mentioned above) and maybe arranged to comprise the polynucleotide operably linked to one ormore expression control sequences, so as to be useful to express therequired enzymes in a host cell, for example a micro-organism asdescribed above.

In some embodiments, the recombinant or genetically modified host cell,as mentioned throughout this specification, may be any micro-organism orpart of a micro-organism selected from the group consisting of fungi(such as members of the genus Saccharomyces), protists, algae, bacteria(including cyanobacteria) and archaea. The bacterium may comprise agram-positive bacterium or a gram-negative bacterium and/or may beselected from the genera Escherichia, Bacillus, Lactobacillus,Rhodococcus, Pseudomonas or Streptomyces. The cyanobacterium may beselected from the group of Synechococcus elongatus, Synechocystis,Prochlorococcus marinus, Anabaena variabilis, Nostoc punctiforme,Gloeobacter violaceus, Cyanothece sp. and Synechococcus sp. Theselection of a suitable micro-organism (or other expression system) iswithin the routine capabilities of the skilled person. Particularlysuitable micro-organisms include Escherichia coli and Saccharomycescerevisiae, for example.

In a related embodiment of the invention, a fatty acid reductase and/ora fatty aldehyde synthetase and/or a fatty acyl transferase and/or analdehyde decarbonylase and/or an acyl-ACP thioesterase and/or a3-ketoacyl-ACP synthase III and/or a branched-chain ketodehydrogenasecomplex or functional variant or functional fragment of any of these maybe expressed in a non-micro-organism cell such as a cultured mammaliancell or a plant cell or an insect cell. Mammalian cells may include CHOcells, COS cells, VERO cells, BHK cells, HeLa cells, Cvl cells, MDCKcells, 293 cells, 3T3 cells, and/or PC12 cells.

The recombinant host cell or micro-organism may be used to express theenzymes mentioned above and a cell-free extract then obtained bystandard methods. Conventional methods and techniques mentioned hereinare explained in more detail, for example, in Sambrook et al. (referencenumber 17, see below).

The identity of amino acid and nucleotide sequences referred to in thisspecification is as set out in Table 7 at the end of the description.The terms “polynucleotide”, “polynucleotide sequence” and “nucleic acidsequence” are used interchangeably herein. The terms “polypeptide”,“polypeptide sequence” and “amino acid sequence” are, likewise, usedinterchangeably herein. Other sequences encompassed by the invention areprovided in the Sequence Listing.

Enzyme Commission (EC) numbers (also called “classes” herein), referredto throughout this specification, are according to the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (NC-IUBMB) in its resource “Enzyme Nomenclature” (1992,including Supplements 6-17) available, for example, athttp://www.chem.qmul.ac.uk/iubmb/enzyme/. This is a numericalclassification scheme based on the chemical reactions catalysed by eachenzyme class (reference 19).

The term “fatty acid reductase complex” indicates an enzyme complexcapable of catalysing the conversion of free fatty acid, fatty acyl-ACPor fatty acyl-CoA to fatty aldehyde. Typically, the complex comprises afatty acid reductase enzyme and a fatty aldehyde synthetase enzyme and afatty acyl transferase enzyme. The term “fatty aldehyde synthetase”indicates an enzyme in class EC 6.2.1.19 capable of catalysing theformation of an acyl-protein thioester from a fatty acid and a protein.The term “fatty acid reductase enzyme” indicates an enzyme in class EC1.2.1.50, the enzyme being capable of catalysing the formation of along-chain aldehyde from a fatty acyl-AMP (fatty acyl-adenosinemonophosphate) or a fatty acyl-CoA. Fatty acyl-AMP is the intermediateformed by the fatty aldehyde synthetase in this coupled reaction. Anexample of a fatty acid reductase is the polypeptide having amino acidsequence SEQ ID NO:1; an example of a fatty aldehyde synthetase is thepolypeptide having amino acid sequence SEQ ID NO:2. Other suitable fattyacid reductase polypeptides have amino acid sequence at least 50%identical to SEQ ID NO:1, e.g., SEQ ID NO:28 or 29; other suitable fattyaldehyde synthetase polypeptides have an amino acid sequence at least50% identical to SEQ ID NO:2, e.g., SEQ ID NO:32 or 33.

The term “fatty acyl transferase” indicates an enzyme in class EC2.3.1.-, capable of catalysing the transfer of the acyl moiety of fattyacyl-ACP, acyl-CoA and other activated acyl donors, to the hydroxylgroup of a serine on the transferase, followed by the conversion of theester to a fatty acid through hydrolysis. An example of a fatty acyltransferase is the polypeptide having amino acid sequence SEQ ID NO:3.Other suitable fatty acyl transferase polypeptides have an amino acidsequence at least 50% identical to SEQ ID NO:3, e.g. SEQ ID NO:30 or 31.

The term “aldehyde decarbonylase” indicates an enzyme in class EC4.1.99.5, capable of catalysing the conversion of fatty aldehyde to ahydrocarbon, for example an alkane, alkene or mixture thereof. Anexample of an aldehyde decarbonylase is the polypeptide having aminoacid sequence SEQ ID NO:4 or an amino acid sequence at least 50%identical to SEQ ID NO:4.

A fatty acid is a carboxylic acid with a long unbranched or branchedaliphatic tail. The fatty acid can comprise saturated fatty acids and/orunsaturated fatty acids containing one, two, three or more double bonds.The one or more fatty acid(s), fatty acyl-ACP or fatty acyl-CoA may, forexample, comprise 4 or more carbon atoms, for example, 8 or more carbonatoms, 10 or more carbon atoms, 12 or more carbon atoms, or 14 or morecarbon atoms. The fatty acid may also comprise, for example, 30 or fewercarbon atoms, for example, 26 or fewer carbon atoms, 25 or fewer carbonatoms, 23 or fewer carbon atoms, or 20 or fewer carbon atoms. Fattyacids may, for example, be derived from triacylglycerols orphospholipids, or may be made de novo by a cell, and/or by mechanismsdescribed elsewhere herein.

In certain embodiments, variants of the polypeptides described hereincan be used. As used herein, a “variant” means a polypeptide in whichthe amino acid sequence differs from the base sequence from which it isderived in that one or more amino acids within the sequence aresubstituted for other amino acids. For example, a variant of SEQ ID NO:1may have an amino acid sequence at least about 50% identical to SEQ IDNO:1, for example, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%identical. The variants and/or fragments are functionalvariants/fragments in that the variant sequence has similar oridenticalfunctional enzyme activity characteristics to the enzyme having thenon-variant amino acid sequence specified herein (and this is themeaning of the term “functional variant” as used throughout thisspecification).

For example, a functional variant of SEQ ID NO:1 has similar oridentical fatty acid reductase characteristics as SEQ ID NO:1, beingclassified in enzyme class EC 1.2.1.50 by the Enzyme Nomenclature ofNC-IUBMB as mentioned above. An example may be that the rate ofconversion by a functional variant of SEQ ID NO:1, in the presence ofnon-variant SEQ ID NO:2, of a free fatty acid to fatty aldehyde may bethe same or similar, for example at least about 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or at least about 100% the rate achieved when usingthe enzyme having amino acid sequence SEQ ID NO:1, in the presence ofnon-variant SEQ ID NO:2. The rate may be improved when using the variantpolypeptide, so that a rate of more than 100% the non-variant rate isachieved. Equivalent analysis of percentage sequence identity andcomparative functional variant activity may, likewise, be made for otherenzymes mentioned herein.

For example, a variant of the fatty acyl transferase SEQ ID NO:3 mayhave an amino acid sequence at least about 50% identical to SEQ ID NO:3,being a functional variant in that it is classified in EC 2.3.1.-; therate of transfer of the acyl moiety of fatty acyl-ACP, acyl-CoA andother activated acyl donors, to the hydroxyl group of a serine on thetransferase, followed by the conversion of the ester to a fatty acidthrough hydrolysis, may be the same or similar, for example at leastabout 60%, 70%, 80%, 90% or 95% the rate achieved when using SEQ IDNO:3.

SEQ ID NOs:28 and 29 may be examples of functional variants of SEQ IDNO:1, as defined herein. SEQ ID NOs:32 and 33 may be examples offunctional variants of SEQ ID NO:2, as defined herein. SEQ ID NOs:30 and31 may be examples of functional variants of SEQ ID NO:3, as definedherein.

The NC-IUBMB classification of the enzymes mentioned herein are, insummary, set out in Table 6 below.

TABLE 6 SEQ ID EC Description of sequence NO number Photorhabdusluminescens LuxC amino acid 1 1.2.1.50 sequence P. luminescens LuxEamino acid sequence 2 6.2.1.19 P. luminescens LuxD amino acid sequence 32.3.1.— Nostoc punctiforme aldehyde decarbonylase amino 4 4.1.99.5 acidsequence Cinnamomum camphora thioesterase amino acid 5 3.1.2.14 sequenceBacillus subtilis KasIII (3-ketoacyl-ACP synthase 6 2.3.1.180 III) aminoacid sequence B. subtilis BCKD subunit E1α amino acid sequence 7 1.2.4.4B. subtilis BCKD subunit E1β amino acid sequence 8 1.2.4.4 B. subtilisBCKD subunit E2 amino acid sequence 9 2.3.1.168 B. subtilis BCKD subunitE3 amino acid sequence 10 1.8.1.4

A functional variant or fragment of any of the above SEQ ID NO aminoacid sequences or genes mentioned herein, therefore, is any amino acidsequence which remains within the same enzyme category (i.e., has thesame EC number) as the non-variant sequences as set out in Table 1.Methods of determining whether an enzyme falls within a particularcategory are well known to the skilled person, who can determine theenzyme category without use of inventive skill Suitable methods may, forexample, be obtained from the International Union of Biochemistry andMolecular Biology.

Amino acid substitutions may be regarded as “conservative” where anamino acid is replaced with a different amino acid with broadly similarproperties. Non-conservative substitutions are where amino acids arereplaced with amino acids of a different type.

By “conservative substitution” is meant the substitution of an aminoacid by another amino acid of the same class, in which the classes aredefined as follows:

Class Amino acid examples Nonpolar: A, V, L, I, P, M, F, W Unchargedpolar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

As is well known to those skilled in the art, altering the primarystructure of a polypeptide by a conservative substitution may notsignificantly alter the activity of that polypeptide because theside-chain of the amino acid which is inserted into the sequence may beable to form similar bonds and contacts as the side chain of the aminoacid which has been substituted out. This is so even when thesubstitution is in a region which is critical in determining thepolypeptide's conformation.

Non-conservative substitutions are possible provided that these do notinterrupt the enzyme activities of the polypeptides, as definedelsewhere herein. The substituted versions of the enzymes must retaincharacteristics such that they remain in the same enzyme class as thenon-substituted enzyme, as determined using the NC-IUBMB nomenclaturediscussed above.

Broadly speaking, fewer non-conservative substitutions than conservativesubstitutions will be possible without altering the biological activityof the polypeptides. Determination of the effect of any substitution(and, indeed, of any amino acid deletion or insertion) is wholly withinthe routine capabilities of the skilled person, who can readilydetermine whether a variant polypeptide retains the enzyme activityaccording to the invention. For example, when determining whether avariant of the polypeptide falls within the scope of the invention(i.e., is a “functional variant or fragment” as defined above), theskilled person will determine whether the variant or fragment retainsthe substrate converting enzyme activity as defined with reference tothe NC-IUBMB nomenclature mentioned elsewhere herein. All such variantsare within the scope of the invention.

Using the standard genetic code, further nucleic acid sequences encodingthe polypeptides may readily be conceived and manufactured by theskilled person, in addition to those disclosed herein. The nucleic acidsequence may be DNA or RNA, and where it is a DNA molecule, it may forexample comprise a cDNA or genomic DNA. The nucleic acid may becontained within an expression vector, as described elsewhere herein.

According to another aspect of the invention, fermentation to producehydrocarbons includes using a recombinant microorganism adapted toexpress at least one of an aldehyde-generating acyl-ACP reductase andfatty aldehyde decarbonylase enzymes. The gene for thealdehyde-generating acyl-ACP reductase and fatty aldehyde decarbonylaseenzymes preferably comes from a cyanobacteria, such as Nostocpunctiforme. Non-limiting examples of the aldehyde-generating acyl-ACPreductase and fatty aldehyde decarbonylase are described in Schirmer A,et al., Microbial biosynthesis of alkanes. Science 329(5991):559-562(2010), the disclosure of which is incorporated by reference in itsentirety. Coexpression aldehyde-generating acyl-ACP reductase and fattyaldehyde decarbonylase enzymes can be generally referred to as NpARAD orNpAR/NpAD.

Embodiments of the invention include variant nucleic acid sequencesencoding the polypeptides described herein. The term “variant” inrelation to a nucleic acid sequence means any substitution of, variationof, modification of, replacement of, deletion of, or addition of one ormore nucleotide(s) from or to a polynucleotide sequence, providing theresultant polypeptide sequence encoded by the polynucleotide exhibits atleast the same or similar enzymatic properties as the polypeptideencoded by the basic sequence. The term includes allelic variants andalso includes a polynucleotide (a “probe sequence”) which substantiallyhybridises to the polynucleotide sequences described herein. Suchhybridisation may occur at or between low and high stringencyconditions. In general terms, low stringency conditions can be definedas hybridisation in which the washing step takes place in a 0.330-0.825M NaCl buffer solution at a temperature of about 40-48 C below thecalculated or actual melting temperature (Tm) of the probe sequence (forexample, about ambient laboratory temperature to about 55 C), while highstringency conditions involve a wash in a 0.0165-0.0330 M NaCl buffersolution at a temperature of about 5-10 C below the calculated or actualTm of the probe sequence (for example, about 65 C). The buffer solutionmay, for example, be SSC buffer (0.15M NaCl and 0.015M tri-sodiumcitrate), with the low stringency wash taking place in 3×SSC buffer andthe high stringency wash taking place in 0.1×SSC buffer. Steps involvedin hybridisation of nucleic acid sequences have been described forexample in Sambrook et al.

Typically, nucleic acid sequence variants have about 55% or more of thenucleotides in common with the nucleic acid sequence of the presentinvention, more typically 60%, 65%, 70%, 80%, 85%, or even 90%, 95%, 98%or 99% or greater sequence identity.

Variant nucleic acids of the invention may be codon-optimised forexpression in a particular host cell.

Sequence identity between amino acid sequences can be determined bycomparing an alignment of the sequences using the Needleman-WunschGlobal Sequence Alignment Tool available from the National Center forBiotechnology Information (NCBI), Bethesda, Md., USA, for example viahttp://blast.ncbi.nlm.nih.gov/Blast.cgi, using default parametersettings (for protein alignment, Gap costs Existence:11 Extension:1).Sequence comparisons and percentage identities mentioned in thisspecification have been determined using this software. When comparingthe level of sequence identity to, for example, SEQ ID NO:1, thistypically should be done relative to the whole length of SEQ ID NO:1(i.e., a global alignment method is used), to avoid short regions ofhigh identity overlap resulting in a high overall assessment ofidentity. For example, a short polypeptide fragment having, for example,five amino acids might have a 100% identical sequence to a five aminoacid region within the whole of SEQ ID NO:1, but this does not provide a100% amino acid identity unless the fragment forms part of a longersequence which also has identical amino acids at other positionsequivalent to positions in SEQ ID NO:1. When an equivalent position inthe compared sequences is occupied by the same amino acid, then themolecules are identical at that position. Scoring an alignment as apercentage of identity is a function of the number of identical aminoacids at positions shared by the compared sequences. When comparingsequences, optimal alignments may require gaps to be introduced into oneor more of the sequences, to take into consideration possible insertionsand deletions in the sequences. Sequence comparison methods may employgap penalties so that, for the same number of identical molecules insequences being compared, a sequence alignment with as few gaps aspossible, reflecting higher relatedness between the two comparedsequences, will achieve a higher score than one with many gaps.Calculation of maximum percent identity involves the production of anoptimal alignment, taking into consideration gap penalties. As mentionedabove, the percentage sequence identity may be determined using theNeedleman-Wunsch Global Sequence Alignment tool, using default parametersettings. The Needleman-Wunsch algorithm was published in J. Mol. Biol.(1970) vol. 48:443-53.

Polypeptide and polynucleotide sequences for use in the methods, vectorsand host cells described herein are shown in the Sequence Listing.

TABLE 7 Identity of sequences included in application SEQ ID NODescription of sequence 1 Photorhabdus luminescens LuxC amino acidsequence 2 P. luminescens LuxE amino acid sequence 3 P. luminescens LuxDamino acid sequence 4 Nostoc punctiforme aldehyde decarbonylase aminoacid sequence 5 Cinnamomum camphora thioesterase amino acid sequence 6Bacillus subtilis KasIII (3-ketoacyl-ACP synthase III) amino acidsequence 7 B. subtilis BCKD subunit E1 amino acid sequence 8 B. subtilisBCKD subunit E1β amino acid sequence 9 B. subtilis BCKD subunit E2 aminoacid sequence 10 B. subtilis BCKD subunit E3 amino acid sequence 11 P.luminescens LuxC codon-optimised nucleotide sequence 12 P. luminescensLuxE codon-optimised nucleotide sequence 13 N. punctiforme aldehydedecarbonylase codon-optimised nucleotide sequence 14 P. luminescens LuxDcodon-optimised nucleotide sequence 15 P. luminescens LuxCDE operoncodon-optimised nucleotide sequence 16 pACYC LuxCDE 17 C. camphorathioesterase codon-optimised nucleotide sequence 18 pETDuet-1thioesterase 19 B. subtilis KasIII codon-optimised nucleotide sequence20 B. subtilis BCKD subunit E1 codon-optimised nucleotide sequence 21 B.subtilis BCKD subunit E1β codon-optimised nucleotide sequence 22 B.subtilis BCKD subunit E2 codon-optimised nucleotide sequence 23 B.subtilis BCKD subunit E3 codon-optimised nucleotide sequence 24KasIII/BCKD operon codon-optimised nucleotide sequence 25 pETDuet-1KasIII/BCKD 26 Amplification primer 27 Amplification primer 28 Vibrioharveyi LuxC amino acid sequence 29 Vibrio fischeri ES114 LuxC aminoacid sequence 30 Vibrio harveyi LuxD amino acid sequence 31 Vibriofischeri MJ11 LuxD amino acid sequence 32 Vibrio harveyi LuxE amino acidsequence 33 Vibrio fischeri ES114 LuxE amino acid sequence

To facilitate a better understanding of embodiments the presentinvention, the following examples of certain aspects of some embodimentsare given. In no way should the following examples be read to limit, ordefine, the entire scope of the invention.

Illustrative Embodiments

The following examples all used solid components obtained as describedbelow.

Biomass Preparation

In this example, various samples of fresh chopped sorghum are mixed witha variety of added components as listed in Table 8 and are stored in asilage bag for about 20 days. The particular additives and respectiveaddition rates are shown in Table 9.

TABLE 8 2011 Experiments WITH ACID Experiment # 1 estimated mass 450 kgsMoisture Content 76% Storage Method Silage bag Yeast Lallemand LiquidYeast bacterial inhibitor Lactrol cellulose to glucose Novozymes CellicCTec2 Chop size 3 mm Result (gallons Ethanol/initial dry metric 50tonne) Days in Storage ~20

TABLE 9 ADDITIVE Rates LACTROL 3.2 g/wet ton Lallemand Stabilized Liquid18 fl oz/wet ton Yeast Novozymes Cellic CTec2 20 fl oz/wet ton 9.3%Concentrated Sulfuric 3.8 L/wet ton Acid

VOC Recovery

The VOCs from the prepared biomass material of this example wererecovered using a GEA SSD™ as the solventless recovery unit. Table 10below provides certain properties of (i) the prepared biomass materialfed into the solventless recovery unit, (ii) the solid component exitingthe solventless recovery unit, and (iii) the operating conditions of thesolventless recovery unit.

TABLE 10 Sample Feed composition Liquid in Feed 80.2% (%) Solidcomponent Liquid in Solid 31.4% component (product) (%) Solid component90 (product) Temperature (F.) Operating Conditions Heater 516Temperature (F.) Feed Rate 5.30 (lb/min.) Evaporation Rate 3.93 (lb/min)Saturation 287 Temperature (F.) Solid component 1.03 production rate(lb/min.) Vapor 428 Temperature at Inlet (F.) Exhaust 370 Temperature(F.) Operating 40 Pressure (psig)

Further Processing: Saccharification

Into a 4 liter bottle was added 2160.02 grams of deionized water and540.12 grams of 40% wt. HESA were mixed to form 8.5% wt. HESA solution.Into a one gallon Parr Instruments C276 autoclave equipped with a DiCompIR probe was placed 433.82 grams of the solid component of Example C.The solid component was estimated to have 289.67 grams of bone driedbiomass (BDBM). The acid solution was gently poured over the wet biomassin the reactor. The reactor contained a mixture comprising approximately9.53% wt. dry biomass in contact with a 7.3% wt. HESA solution (based onthe total reactor content).

The reaction mixture was heated to 120 degrees C. and held for thestated period of time. The reactor content was stirred initially at 100rpm, but as the reaction heats to 120° C. the contents thin and the stirrate is increased to 250 then 400 rpm. The reactor was held at 120° C.for 1 hour. The heating was discontinued. The reactor was purged with aslow nitrogen stream for a few minutes to eliminate any sulfur dioxidein the gas cap. The reactor was cooled to room temperature and purgedonce more with nitrogen.

The reactor content was transferred to a Buchner funnel and vacuumfiltered over Whatman 541 hardened ashless 185 mm filter paper. As muchliquid as possible was removed from the reactor content. The cumulativeweight of the filtrate and liquids removed was obtained. The filtratewas then analyzed by HPLC and the recovery of materials from the biomasscalculated by comparison to the amount of the precursors in present inthe biomass. The % of glucose recovered was 11.3%, based on thetheoretical amount of glucose available in the biomass. The % of xyloserecovered was 91%, based on the theoretical amount of xylose availablein the biomass.

The treated sample was further subject to enzymatic hydrolysis. 144grams of the material from HESA treatment were washed 3 times with 500mL deionized water. After the first wash, the pH of the material wasadjusted to 10. The liquid was then drained, and water was added, andthe pH was adjusted to 5.6. In 1 L of water with about 144 grams ofwashed material, 50 grams of CTEC2 cellulase were added. The solutionwas shaken at 53 degrees Celsius for 3 days at which time the contentswere measured to be: Cellobiose: 1.93 g/L, Glucose: 52.6 g/L, Xylose:6.12 g/L, Arabinose: 0 g/L, Glycerol: 1.4 g/L, Acetic Acid: 0.92 g/L,Ethanol: 0.0 g/L.

Fermentation

The hydrolysis mixture (or hydrolysate) was then fermented, where thehydrolysate was added to the media growing various E. coli cultures toreplace the glucose content. The E. coli cultures were adapted toco-express the fatty acid reductase, fatty aldehyde synthetase, fattyacyl transferase, and aldehyde decarbonylase enzymes (CEDDEC) or NpARAD.E. coli host cells without these exogenous genes (BL21) were also grownfor reference.

In general, E. coli BL21*(DE3) cells carrying pACYCDuet NpAR/AD,pACYCDuet CEDDEC or no vector controls were grown from glycerol stocksat 37 degrees C., with constant shaking (225 rpm) overnight in LB mediawith 34 ug/mL chloramphenicol as selectable marker. Chloramphenicol wasomitted from E. coli BL21*(DE3) control cells. 200 uL of overnightstarter cultures were used to inoculate 20 mL of the following media,containing antibiotic as indicated, and prepared as follows: modifiedminimal media, without yeast extract*and with the 3% glucose carbonsource replaced with filter sterilised hydrolysis mix to a final, totalcarbon (cellobiose+glucose+xylose+glycerol+acetic acid) concentration of1%. This can be referred to as the standard LB medium. In addition, E.coli BL21*(DE3) cells carrying pACYCDuet CEDDEC were inoculated intomodified minimal media with yeast extract (MYE) and with a 3% glucosecarbon source as a positive control. Three replicates of each treatmentwere grown. This can be referred to as the LB-MYE medium.

Construction of FAR/NpAD (CEDDEC) Plasmids

The amino acid sequences listed in Table 2 below were reverse translatedand codon-optimised for expression in E. coli, providing the nucleicacid sequences also shown in Table 2:

TABLE 11 Codon-optimised GenBank** nucleic acid accession numberSequence name SEQ ID NO SEQ ID NO AAD05355.1 Fatty acid reductase 1 11AAD05359.1 LuxE E 2 12 P19197.1 LUXD1_PHOLU 3 13 **The sequences can beretrieved from GenBank at http://www.ncbi.nlm.nih.gov/genbank. GenBankis the NIH genetic sequence database. Genbank is located at the NationalCenter for Biotechnology Information, U.S. National Library of Medicine,8600 Rockville Pike, Bethesda MD, 20894 USA.

Codon-optimised luxC, luxE and luxD genes for E. Coli were synthesisedin a three-gene operon (SEQ ID NO:15) inserted into pACYCDuet-1(commercially obtainable from Merck, the final construct having sequenceSEQ ID NO:16) and subsequently digested with the restriction enzymesNcoI and NotI (commercially obtainable) and ligated into pCDFDuet-1 MCS1(commercially obtainable from Merck).

The Genomic DNA was extracted from N. punctiforme using the FAST-DNASPIN Kit (commercially obtainable by MP Biomedicals). Cultures werecentrifuged for 2 min, 4500 rpm, 4 C and 120 mg of the pellet wasre-suspended in 1 ml of buffer Cell Lysis/DNA Solubilizing Solution(CLS-Y). Samples were homogenized with a MP Biomedicals FastPrep-24(FASTPREP is a trademark) instrument using lysing matrix A (also MPBiomedicals) for 40 sec at a speed setting of 6.0 m/s. All subsequentsteps were carried out according to the manufacturer's instructions.After this procedure, the genomic DNA was further purified byphenol-chloroform extraction (using a tris(hydroxymethyl)aminomethane)

pH7.5-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution),followed by DNA precipitation using ethanol and sodium acetate. Thefinal DNA samples were adjusted (using water) to a concentration of 8nanograms per microliter (ng/μl). The gene encoding NpAD (aldehydedecarbonylase) was amplified with PHUSION High-Fidelity DNA Polymerase(PHUSION is a trademark, commercially obtainable from New EnglandBiolabs), using 8 ng of cyanobacterial genomic DNA as template.

Primers used were CATATGCAGCAGCTTACAGACCAAT (SEQ ID NO:26) andCTCGAGTTAAGCACCTATGAGTCCGTAGG (SEQ ID NO:27), allowing direct cloninginto MCS2 (MCS is an abbreviation for Multiple Cloning Site) using NdeIand XhoI sites (underlined).

Plasmids were transformed into TOP10 competent E. coli cells(commercially obtainable from Invitrogen) using the manufacturesprotocol (as described above for Expression of recombinant enzymes in E.coli), purified using the Qiagen miniprep kit (purified plasmids) andinsertions were investigated by polymerase chain reaction (PCR) orrestriction digest. The nucleic acid sequence SEQ ID NO:13, encodingNpAD, was confirmed to be present in pACYCDuet-1 luxCED and pCDFDuet-1luxCED by DNA sequencing (commercially obtainable from Geneservice,U.K.) of purified plasmids.

Similar techniques were used to construct the NpARAD expression plasmid.E. coli cultures containing the FAR/NpAD (CEDDEC) expression plasmid,NpARAD expression plasmid, or no expression plasmid (BL21) wereinoculated and grown in the standard LB media for at least 6 hours. Inaddition, an E. coli cultures containing the FAR/NpAD (CEDDEC) was alsogrown in LB-MYE media for at least 6 hours. Table 12 below shows theoptical reading at OD600 nm at various time points during the growth ofthe bacteria cultures.

TABLE 12 hr post CEDDEC inoculation CEDDEC NpARAD BL21 (MYE) 2 0.24 0.240.33 0.03 3.5 0.95 0.93 1.13 0.31 6 0.73

FIG. 5 shows the growth curve of these different E. coli cultures:CEDDEC, NpARAD, BL21, and CEDDEC (MYE), which is E. coli containingCEDDEC expression plasmid grown in LB-MYE medium. As shown, the growthof E. coli cultures containing the FAR/NpAD (CEDDEC) and NpARADexpression plasmids were quite robust, essentially the same as thestandard culture without any expression plasmid. This shows that theglucose in the hydrolysis mixture was available to the microorganismsand further that the hydrolysis mixture was not toxic to themicroorganisms.

While the cultures were not harvested for hydrocarbon extraction anddetection, based on the demonstration by PCT/EP2013/053600 ofhydrocarbon production by E. coli cultures containing the FAR/NpAD(CEDDEC) and NpARAD expression plasmids, it is expected that thecultures of this example would provide a similar outcome, particularlyin light of such robust growth. The hydrocarbons can be extracted anddetected by the following method.

8 ml of bacterial culture can be mixed with 8 ml of ethyl acetate andincubated for 2 hours at room temperature (about 20° C.) and 480 rpm tofacilitate hydrocarbon extraction. After extraction, samples can becentrifuged at room temperature (about 20° C.), 700× gravitation for 5minutes to cause phase separation, and 6 ml of the top phase can betransferred into a fresh vial. The ethyl acetate can be dried under astream of nitrogen and subsequently the residue can be dissolved in 225ml dichloromethane (DCM). Separation and identification of hydrocarbonsand volatile compounds can be performed using a TraceGasChromatography-Mass spectrometer (GC/MS) 2000 (Thermo Finnigan)equipped with a ZB1-MS column (commercially obtainable from Zebron).

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

We claim:
 1. A method for processing a biomass material comprising: (i)introducing a biomass material to a compartment of a solventlessrecovery system, wherein the biomass material contains one or morevolatile organic compounds; (ii) contacting the biomass material with asuperheated vapor stream in the compartment to vaporize at least aportion of an initial liquid content in the biomass material, saidsuperheated vapor stream comprising at least one volatile organiccompound; (iii) separating a vapor component and a solid component fromthe heated biomass material, said vapor component comprising at leastone volatile organic compound; retaining at least a portion of the gascomponent for use as part of the superheated vapor stream; (iv)discharging the solid component from the solventless recovery system,wherein the solid component comprises a lignocellulosic material; (v)generating at least one fermentable sugar from the lignocellulosicmaterial; and (vi) contacting the at least one fermentable sugar with amicroorganism capable of using the at least one fermentable sugar togenerate a hydrocarbon.
 2. The method of claim 1, wherein themicroorganism is adapted to express at least one of a fatty acidreductase, a fatty aldehyde synthetase, a fatty acyl transferase, and analdehyde decarbonylase.
 3. The method of claim 2, wherein themicroorganism comprises at least one nucleic acid encoding one or moreof the amino acid sequences selected from the group consisting of SEQ IDNO:1 to SEQ ID NO:4.
 4. The method of claim 3, wherein the microorganismis a yeast or a bacterium.
 5. The method of claim 4, wherein the yeastis Saccharomyces cerevisiae and the bacterium is Eschericia coli.
 6. Themethod of claim 2, wherein the microorganism is further adapted toexpress an acyl-ACP thioesterase.
 7. The method of claim 6, wherein themicroorganism is a genetically modified microorganism geneticallymodified to express an exogenous acyl-ACP thioesterase.
 8. The method ofclaim 7, wherein the microorganism comprises at least one nucleic acidencoding the amino acid sequence of SEQ ID NO:5.
 9. The method of claim2, wherein the microorganism is further adapted to express a3-ketoacyl-ACP synthase III.
 10. The method of claim 9, wherein the hostcell comprises at least one nucleic acid encoding the amino acidsequence of SEQ ID NO:6.
 11. The method of claim 2, wherein themicroorganism is further adapted to express a branched-chainketodehydrogenase complex.
 12. The method of claim 11, wherein themicroorganism comprises at least one nucleic acid encoding one or moreof the amino acid sequences selected from the group consisting of SEQ IDNO:7 to SEQ ID NO:10.
 13. The method of claim 1, wherein themicroorganism is adapted to express an aldehyde-generating acyl-ACPreductase and fatty aldehyde decarbonylase.
 14. The method of claim 1wherein the biomass material introduced to the compartment is obtainedfrom a fermentation process of a harvested crop.
 15. The method of claim14 wherein the crop is selected from the group consisting of sorghum,sugar cane, corn, tropical corn, sugar beet, energy cane, and anycombination thereof.
 16. The method of claim 1 wherein the compartmentcomprises a cylindrical body in a shape of a loop within which thesuperheated vapor stream flows.
 17. The method of claim 1 wherein theseparating step is achieved using a cyclone separating component coupledto the compartment, wherein the cyclone separating component isconfigured to discharge the separated solid component from thecompartment.
 18. The method of claim 1 the biomass is generated byadding to the biomass at least one additive added, wherein said at leastone additive comprise a microbe, and optionally, an acid and/or anenzyme; and storing the prepared biomass material for at least about 24hours in a storage facility to allow for the production of at least onevolatile organic compound from at least a portion of the sugar.
 19. Amethod for processing a biomass material comprising: contacting a solidcomponent of a biomass material with a solution adapted to facilitatesaccharification, and contacting the at least one fermentable sugar witha microorganism capable of using the at least one fermentable sugar togenerate a hydrocarbon. wherein the solid component is generated by amethod comprising: introducing a biomass material to a compartment of asolventless recovery system, wherein the biomass material contains oneor more volatile organic compounds; contacting the biomass material witha superheated vapor stream in the compartment to vaporize at least aportion of an initial liquid content in the biomass material, saidsuperheated vapor stream comprising at least one volatile organiccompound; separating a vapor component and a solid component from theheated biomass material, said vapor component comprising at least onevolatile organic compound; retaining at least a portion of the gascomponent for use as part of the superheated vapor stream; dischargingthe solid component from the solventless recovery system.